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  • Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy. 20008564

    Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.
    Tipo de documento:
    Referencia
    Referencia del producto:
    12-371
    Nombre del producto:
    Normal Mouse IgG

  • Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons. 19081133

    Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca(2+)) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca(2+) signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP(3)R-mediated intracellular Ca(2+) responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca(2+) signaling and its related functions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extrac ... 12605860

    We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotinylated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay procedure was validated using recombinant PI 3-kinase gamma with diC8PtdIns(4,5)P(2) as substrate and general receptor for phosphoinositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P(3)-specific probe. This PI 3-kinase assay was robust, was suitable for high-throughput screening platforms, and delivered expected IC(50) values for reference compounds. The approach is adaptable to a wide range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was measured using the same reagents but with diC8PtdIns(3,4,5)P(3) as substrate. PtdIns(3,4,5)P(3) present in lipid extracts of Swiss 3T3 and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity. The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1 (which binds PtdIns(3,4)P(2) and not PtdIns(3,4,5)P(3)). This system was used to monitor the accumulation of PtdIns(3,4)P(2) in Swiss 3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-characterized protein domains with appropriate phosphoinositide-binding specificity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    14-558
    Nombre del producto:
    PI3 Kinase (p120γ), 20 µg

  • CG0006, a novel histone deacetylase inhibitor, induces breast cancer cell death via histone-acetylation and chaperone-disrupting pathways independent of ER status. 21184271

    We previously reported that CG0006, a novel hydroxamate-based pan-histone deacetylase inhibitor (HDACI), suppresses the growth of human cancer cells. Here, we tested the ability of CG0006 to inhibit breast cancer cell proliferation in relation to estrogen receptor (ER) status, and examined changes in the expression of cell-cycle regulatory proteins. CG0006 effects on the proliferation of multiple human cancer cell lines were tested using MTT and MTS assays. Changes in estrogen-signaling proteins and cell-cycle regulatory proteins were examined by western blotting and quantitative RT-PCR, and cell-cycle effects were tested using flow cytometry. CG0006 increased histone H3 and H4 acetylation, up-regulated p21 protein, and promoted cell-cycle arrest, inducing G(2)/M-phase accumulation in ER-positive MCF7 cells, and G(1)- and G(2)/M-phase accumulation in ER-negative MDA-MB-231 cells. In both cell types, CG0006 treatment (1 μM) reduced the levels of the estrogen-signaling proteins ERα and cyclin D1, and promoted massive degradation of cell-cycle regulatory proteins. CG0006 down-regulated the histone deacetylase HDAC6 at the protein level in association with a subsequent increase in Hsp90 and α-tubulin acetylation. HDAC6 depletion using small interfering RNA produced a protein-degradation phenotype similar to that of CG0006 treatment. These findings suggest that CG0006 inhibits breast cancer cell growth by two different pathways: a histone acetylation-dependent pathway, and a non-epigenetic pathway that disrupts chaperone function.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-748

  • Mechanistic insight into the ability of American ginseng to suppress colon cancer associated with colitis. 20729391

    We have recently shown that American ginseng (AG) prevents and treats mouse colitis. Because both mice and humans with chronic colitis have a high colon cancer risk, we tested the hypothesis that AG can be used to prevent colitis-driven colon cancer. Using the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of ulcerative colitis, we show that AG can suppress colon cancer associated with colitis. To explore the molecular mechanisms of the anticancer effects of AG, we also carried out antibody array experiments on colon cells isolated at a precancerous stage. We found there were 82 protein end points that were either significantly higher (41 proteins) or significantly lower (41 proteins) in the AOM + DSS group compared with the AOM-alone (control) group. In contrast, there were only 19 protein end points that were either significantly higher (10 proteins) or significantly lower (9 proteins) in the AOM + DSS + AG group compared with the AOM-alone (control) group. Overall, these results suggest that AG keeps the colon environment in metabolic equilibrium when mice are treated with AOM + DSS and gives insight into the mechanisms by which AG protects from colon cancer associated with colitis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-1224
    Nombre del producto:
    Anti-PP6C Antibody

  • Neuronal death resulting from targeted disruption of the Snf2 protein ATRX is mediated by p53. 19020049

    ATRX, a chromatin remodeling protein of the Snf2 family, participates in diverse cellular functions including regulation of gene expression and chromosome alignment during mitosis and meiosis. Mutations in the human gene cause alpha thalassemia mental retardation, X-linked (ATR-X) syndrome, a rare disorder characterized by severe cognitive deficits, microcephaly and epileptic seizures. Conditional inactivation of the Atrx gene in the mouse forebrain leads to neonatal lethality and defective neurogenesis manifested by increased cell death and reduced cellularity in the developing neocortex and hippocampus. Here, we show that Atrx-null forebrains do not generate dentate granule cells due to a reduction in precursor cell number and abnormal migration of differentiating granule cells. In addition, fewer GABA-producing interneurons are generated that migrate from the ventral telencephalon to the cortex and hippocampus. Staining for cleaved caspase 3 demonstrated increased apoptosis in both the hippocampal hem and basal telencephalon concurrent with p53 pathway activation. Elimination of the tumor suppressor protein p53 in double knock-out mice rescued cell death in the embryonic telencephalon but only partially ameliorated the Atrx-null phenotypes at birth. Together, these findings show that ATRX deficiency leads to p53-dependent neuronal apoptosis which is responsible for some but not all of the phenotypic consequences of ATRX deficiency in the forebrain.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1583
    Nombre del producto:
    Anti-Neuropeptide Y Antibody

  • A novel TNFR1-triggered apoptosis pathway mediated by class IA PI3Ks in neutrophils. 21478427

    The most common form of neutrophil death is apoptosis. In the present study, we report surprising differences in the molecular mechanisms used for caspase activation between FAS/CD95-stimulated and TNF receptor 1 (TNFR1)-stimulated neutrophils. Whereas FAS-induced apoptosis was followed by caspase-8 activation and required Bid to initiate the mitochondrial amplification loop, TNF-α-induced apoptosis involved class IA PI3Ks, which were activated by MAPK p38. TNF-α-induced PI3K activation resulted in the generation of reactive oxygen species, which activated caspase-3, a mechanism that did not operate in neutrophils without active NADPH oxidase. We conclude that in neutrophils, proapoptotic pathways after TNFR1 stimulation are initiated by p38 and PI3K, but not by caspase-8, a finding that should be considered in anti-inflammatory drug-development strategies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-195

  • Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver. 18182619

    Neuropilin-2 (NRP2) is a high-affinity kinase-deficient receptor for vascular endothelial growth factor (VEGF) and semaphorin 3F. We investigated its function in human colorectal cancers.Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA (shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for activation of the VEGFR1 pathway by immunoblotting. Soft agar assays, Annexin V staining, and Boyden chamber assays were used to examine anchorage-independent growth, apoptosis in response to hypoxia, and cell migration/invasion, respectively, in HCT-116 transfectants. Tumor growth and metastasis were analyzed in mice (groups of 10) injected with shRNA-expressing HCT-116 cells. The effect of in vivo targeting of NRP2 by small interfering RNA (siRNA) on the growth of hepatic colorectal tumors derived from luciferase-expressing HCT-116 cells was assessed by measuring changes in bioluminescence and final tumor volumes. All statistical tests were two-sided.NRP2 expression was substantially higher in tumors than in adjacent mucosa. HCT-116 transfectants with reduced NRP2 levels had reduced VEGFR1 signaling, but proliferation was unchanged. Anchorage-independent growth, survival under hypoxic conditions, and motility/invasiveness were also reduced. In vivo, HCT-116 transfectants with reduced NRP2 demonstrated decreased tumor growth, fewer metastases, and increased apoptosis compared with control cells. Hepatic colorectal tumors in mice treated with NRP2 siRNAs were statistically significantly smaller than those in mice treated with control siRNAs (at 28 days after implantation, mean control siRNAs = 420 mm3, mean NRP2 siRNAs = 36 mm3, NRP2 vs control: difference = 385 mm3, 95% confidence interval = 174 mm3 to 595 mm3, P = .005).NRP2 on colorectal carcinoma cells is important for tumor growth and is a potential therapeutic target in human cancers where it is expressed.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-758
    Nombre del producto:
    Anti-phospho-Flt-1 (Tyr1213) Antibody

  • Increased KIT signalling with up-regulation of cyclin D correlates to accelerated proliferation and shorter disease-free survival in gastrointestinal stromal tumours (GIS ... 18729075

    Gastrointestinal stromal tumours (GISTs) with deletions in KIT exon 11 are characterized by higher proliferation rates and shorter disease-free survival times, compared to GISTs with KIT exon 11 point mutations. Up-regulation of cyclin D is a crucial event for entry into the G1 phase of the cell cycle, and links mitogenic signalling to cell proliferation. Signalling from activated KIT to cyclin D is directed through the RAS/RAF/ERK, PI3K/AKT/mTOR/EIF4E, and JAK/STATs cascades. ERK and STATs initiate mRNA transcription of cyclin D, whereas EIF4E activation leads to increased translation efficiency and reduced degradation of cyclin D protein. The aim of the current study was to analyse the mRNA and protein expression as well as protein phosphorylation of central hubs of these signalling cascades in primary GISTs, to evaluate whether tumours with KIT exon 11 deletions and point mutations differently utilize these pathways. GISTs with KIT exon 11 deletions had significantly higher mitotic counts, higher proliferation rates, and shorter disease-free survival times. In line with this, they had significantly higher expression of cyclin D on the mRNA and protein level. Furthermore, there was a significantly higher amount of phosphorylated ERK1/2, and a higher protein amount of STAT3, mTOR, and EIF4E. PI3K and phosphorylated AKT were also up-regulated, but this was not significant. Ultimately, GISTs with KIT exon 11 deletions had significantly higher phosphorylation of the central negative cell-cycle regulator RB. Phosphorylation of RB is accomplished by activated cyclin D/CDK4/6 complex, and marks a central event in the release of the cell cycle. Altogether, these observations suggest increased KIT signalling with up-regulation of cyclin D as the basis for the unfavourable clinical course in GISTs with KIT exon 11 deletions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-157

  • Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise. 15949699

    Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1694