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  • Prenatal plus postnatal exposure to Di(n-Butyl) phthalate and/or flutamide markedly reduces final sertoli cell number in the rat. 20392824

    Androgens may be important regulators of Sertoli cell (SC) proliferation perinatally, with implications for the testicular dysgenesis syndrome (TDS) hypothesis. Fetal exposure of rats to 500 mg/kg . d di(n-butyl) phthalate (DBP) reduces fetal testosterone production and SC number at birth, but SC number recovers to normal by postnatal d (Pnd)25. It is unclear when and how SC proliferation is affected prenatally by DBP exposure or when and how postnatal compensation occurs. This study addressed these questions and investigated whether continued maternal exposure to DBP or to flutamide from Pnd1-Pnd15 could prevent SC number compensation, because this would have implications for how sperm counts might be lowered in TDS. DBP exposure attenuated SC proliferation by 7-18% throughout embryonic d (e)15.5-e21.5 (P < 0.05 at e21.5). After birth, SC proliferation increased significantly (>1.5-fold) between Pnd6 and Pnd10 in prenatally DBP-exposed animals, explaining the compensation. Continued maternal administration of DBP after birth attenuated (19% reduction) SC number compensation at Pnd25 and maternal administration of flutamide (100 mg/kg . d) to prenatally DBP-exposed animals was even more effective (42% reduction), suggesting the postnatal compensatory increase in SC proliferation after prenatal DBP exposure is androgen dependent. SC maturation (Pnd25) was unaffected, based on analysis of expression of key proteins, but lumen formation/expansion was attenuated in parallel with treatment-induced reduction in SC number. Our results provide further evidence that perinatal SC proliferation is androgen dependent and, importantly, show that similar exposure of mothers to antiandrogenic chemicals before birth and during lactation reduces final SC number, with implications for the origin of low sperm counts in TDS.
    Document Type:
    Reference
    Product Catalog Number:
    MAB318
    Product Catalog Name:
    Anti-Tyrosine Hydroxylase Antibody, clone LNC1 - (Anti-Tyrosine Hydroxylase Antibody, clone LNC1)

  • Androgen receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters. 24201806

    The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. However, the molecular mechanisms that underlie the ligand-independent apoptosis, including the activity of AR in testicular stem cells, are not completely understood. In the present study, we generated induced pluripotent stem cells (iPSCs) from bovine testicular cells by electroporation of octamer-binding transcription factor 4 (OCT4). The cells were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4, which maintained and stabilized the expression of stemness genes and pluripotency. The iPSCs were used to assess the apoptosis activity following exposure to phthalate esters, including di (2-ethyhexyl) phthalates, di (n-butyl) phthalate, and butyl benzyl phthalate. Phthalate esters significantly reduced the expression of AR in iPSCs and induced a higher ratio of BAX/BCL-2, thereby favoring apoptosis. Phthalate esters also increased the expression of cyclin-dependent kinase inhibitor 1 (p21(Cip1)) in a p53-dependent manner and enhanced the transcriptional activity of p53. The forced expression of AR and knockdown of p21(Cip1) led to the rescue of the phthalate-mediated apoptosis. Overall, this study suggests that testicular iPSCs are a useful system for screening the toxicity of environmental disruptors and examining their effect on the maintenance of stemness and pluripotency, as well as for identifying the iPSC signaling pathway(s) that are deregulated by these chemicals.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Transcriptional and post-transcriptional upregulation of p27 mediates growth inhibition of isorhapontigenin (ISO) on human bladder cancer cells. 29409027

    There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™ - (EZ-ChIP™)

  • z-Leucinyl-leucinyl-norleucinal induces apoptosis of human glioblastoma tumor-initiating cells by proteasome inhibition and mitotic arrest response. 19861404

    Gamma-secretase inhibitors have been proposed as drugs able to kill cancer cells by targeting the NOTCH pathway. Here, we investigated two of such inhibitors, the Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle) and the N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), to assess whether they were effective in killing human glioblastoma tumor-initiating cells (GBM TIC) in vitro. We found that only LLNle was able at the micromolar range to induce the death of GBM TICs by apoptosis. To determine the cellular processes that were activated in GBM TICs by treatment with LLNle, we analyzed the amount of the NOTCH intracellular domain and the gene expression profiles following treatment with LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, beside inhibiting the generation of the NOTCH intracellular domain, also induces proteasome inhibition, proteolytic stress, and mitotic arrest in these cells by repressing genes required for DNA synthesis and mitotic progression and by activating genes acting as mitotic inhibitors. DNA content flow cytometry clearly showed that cells treated with LLNle undergo arrest in the G(2)-M phases of the cell cycle. We also found that DAPT and L-685,458, another selective Notch inhibitor, were unable to kill GBM TICs, whereas lactacystin, a pure proteasome inhibitor, was effective although at a much less extent than LLNle. These data show that LLNle kills GBM TIC cells by inhibiting the proteasome activity. We suggest that LLNle, being able to target two relevant pathways for GBM TIC survival,-05-have a potential therapeutic value that deserves further investigation in animal models.
    Document Type:
    Reference
    Product Catalog Number:
    04-262
    Product Catalog Name:
    Anti-Ubiquitinylated proteins Antibody, clone FK1 - (Anti-Ubiquitinylated proteins Antibody, clone FK1)

  • Reduction of β-amyloid deposits by γ-secretase inhibitor is associated with the attenuation of secondary damage in the ipsilateral thalamus and sensory functional improve ... 20683452

    Abnormal β-amyloid (Aβ) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aβ deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aβ accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aβ, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aβ burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aβ deposits are associated with the secondary thalamic damage. Reduction of thalamic Aβ by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Tyrosine phosphorylation of HPK1 by activated Src promotes ischemic brain injury in rat hippocampal CA1 region. 18498770

    Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that HPK1 is involved in c-Jun NH2-terminal kinase (JNK) signaling pathway by sequential activation of MLK3-MKK7-JNK3 after cerebral ischemia. Here, we used 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) and MK801 to investigate the events upstream of HPK1 in ischemic brain injury. Immunoprecipitation and immunoblot results showed that PP2 and MK801 significantly decreased the activation of Src, HPK1, MLK3, JNK3 and c-Jun, respectively, during ischemia/reperfusion. Histology and TUNEL staining showed PP2 or MK801 protects against neuron death after brain ischemia. We speculate that this unique signaling pathway through the tyrosine phosphorylation of HPK1 promotes ischemic brain injury by activated Src via N-methyl-d-aspartate receptor and, ultimately, the activation of the MLK3-MKK7-JNK3 pathway after cerebral ischemia.
    Document Type:
    Reference
    Product Catalog Number:
    05-184

  • Synthesis and biodistribution of (11)C-GW7845, a positron-emitting agonist for peroxisome proliferator-activated receptor-{gamma}. 16204723

    The goal of this study was to synthesize and evaluate in vivo the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist (11)C-GW7845 ((S)-2-(1-carboxy-2-{4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl}ethylamino)benzoic acid methyl ester) ((11)C-compound 1). PPARgamma is a member of a family of nuclear receptors that plays a central role in the control of lipid and glucose metabolism. Compound 1 is an analog of tyrosine (inhibitor constant, 3.7 nmol/L), which is an inhibitor of experimental mammary carcinogenesis. METHODS: Protection of the carboxylic acid moiety of compound 1 was effected by treatment with N,N-dimethylformamide di-tert-butyl acetal to provide compound 2. Hydrolysis of the carbomethoxy group of compound 2 provided the benzoic acid (compound 3) that served as an immediate precursor to radiolabeling. Compound 3 underwent treatment with (11)C-methyl iodide followed by high-performance liquid chromatography to produce a radioactive peak sample that coeluted with a standard sample of compound 1. Analysis of biodistribution was undertaken by injecting male CD-1 mice via the tail vein with 6.03 MBq (163 microCi, 2.55 microg/kg) of (11)C-compound 1. To determine the tumor uptake of the radiotracer, 6 female SCID mice bearing MCF-7 xenografts were injected via the tail vein with 10.5 MBq (283 microCi, 0.235 microg/kg) of (11)C-compound 1. RESULTS: (11)C-Compound 1 was synthesized at an 8% radiochemical yield in 29 min with an average specific radioactivity of 1,222 GBq/micromol (33,024 mCi/micromol; n = 6) at the end of synthesis. Spleen (target)-to-muscle uptake and tumor-to-muscle uptake ratios were 3.1 and 1.5, respectively, but this uptake could not be blocked with unlabeled compound 1 at 2 mg/kg. CONCLUSION: Further structural modification, perhaps to generate a less lipophilic tyrosine analog, will be necessary to enable receptor-mediated PPARgamma imaging by this class of agents.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3872
    Product Catalog Name:
    Anti-PPAR γ Antibody, isoform 1&2 - (Anti-PPAR γ Antibody, isoform 1&2)

  • Presenilin 1/gamma-secretase is associated with cadmium-induced E-cadherin cleavage and COX-2 gene expression in T47D breast cancer cells. 18791180

    Cadmium is a heavy metal that has multiple toxic effects on human health and has been classified as a human carcinogen. E-cadherin is a major target of cadmium; however, the roles of E-cadherin and cadmium and the mechanisms of tumor progression remain to be defined. Here, we demonstrate that cadmium increases E-cadherin processing via a gamma-secretase in the T47D breast cancer cell lines. This presenilin 1 (PS1)/gamma-secretase-dependent cleavage of E-cadherin was accompanied by changes in reactive oxygen species or calcium. E-cadherin cleavage was blocked by a PS1 dominant-negative mutant, gamma-secretase inhibitors [N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and L-685,486], antioxidants (N-acetylcysteine and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride), or a calcium chelating drug 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester. Immunofluorescence analysis confirmed the disappearance of E-cadherin staining at the cell surface. Those inhibitors attenuated cadmium-induced cytotoxicity. Additionally, cadmium treatment increased cell motility and invasion ability, which was abated by DAPT. Interestingly, cyclooxygenase-2 (COX-2) expression induced by cadmium was also inhibited by DAPT. The cadmium-induced cell motility and invasion ability were inhibited by a COX-2 inhibitor, NS398. Our data indicate a novel molecular mechanism that links cytotoxicity of cadmium and disrupted E-cadherin processing to adherens junctions; cadmium induces COX-2 expression via gamma-secretase, which increases cell motility and invasion ability. Understanding the downstream signaling cascades of cadmium that promote tumor progression might be a key to the development of novel therapeutic strategies.
    Document Type:
    Reference
    Product Catalog Number:
    ECM508
    Product Catalog Name:
    QCM Chemotaxis Cell Migration Assay, 24-well (8 µm), colorimetric - (QCM Chemotaxis Cell Migration Assay, 24-well (8 µm), colorimetric)

  • Macrophage migration inhibitory factor up-regulates matrix metalloproteinase-9 and -13 in rat osteoblasts. Relevance to intracellular signaling pathways. 11751895

    Neutral matrix metalloproteinases (MMPs) play an important role in bone matrix degradation accompanied by bone remodeling. We herein show for the first time that macrophage migration inhibitory factor (MIF) up-regulates MMP-13 (collagenase-3) mRNA of rat calvaria-derived osteoblasts. The mRNA up-regulation was seen at 3 h in response to MIF (10 microg/ml), reached the maximum level at 6-12 h, and returned to the basal level at 36 h. MMP-13 mRNA up-regulation was preceded by up-regulation of c-jun and c-fos mRNA. Tissue inhibitor of metalloproteinase (TIMP)-1 and MMP-9 (92-kDa type IV collagenase) were also up-regulated, but to a lesser extent. The MMP-13 mRNA up-regulation was significantly suppressed by genistein, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Similarly, a selective mitogen-activated protein kinase (MAPK) kinase (MEK)1/2 inhibitor (PD98059) and c-jun/activator protein (AP)-1 inhibitor (curcumin) suppressed MMP-13 mRNA up-regulation induced by MIF. The mRNA levels of c-jun and c-fos in response to MIF were also inhibited by PD98059. Consistent with these results, MIF stimulated phosphorylation of tyrosine, autophosphorylation of Src, activation of Ras, activation of extracellular signal-regulated kinases (ERK) 1/2, a MAPK, but not c-Jun N-terminal kinase or p38, and phosphorylation of c-Jun. Osteoblasts obtained from calvariae of newborn JunAA mice, defective in phosphorylation of c-Jun, or newborn c-Fos knockout (Fos -/- ) mice, showed much less induction of MMP-13 with the addition of MIF than osteoblasts obtained from wild-type or littermate control mice. Taken together, these results suggest that MIF increases the MMP-13 mRNA level of rat osteoblasts via the Src-related tyrosine kinase-, Ras-, ERK1/2-, and AP-1-dependent pathway.
    Document Type:
    Reference
    Product Catalog Number:
    17-218
    Product Catalog Name:
    Ras Activation Assay Kit - (Ras Activation Assay Kit)

  • The transcription factor hairy/E(spl)-related 2 induces proliferation of neural progenitors and regulates neurogenesis and gliogenesis. 25446033

    The study of molecular regulation in neural development provides information to understand how diverse neural cells are generated. It also helps to establish therapeutic strategies for the treatment of neural degenerative disorders and brain tumors. The Hairy/E(spl) family members are potential targets of Notch signaling, which is fundamental to neural cell maintenance, cell fate decisions, and compartment boundary formation. In this study, we isolated a zebrafish homolog of Hairy/E(spl), her2, and showed that this gene is expressed in neural progenitor cells and in the developing nervous system. The expression of her2 required Notch activation, as revealed by a Notch-defective mutant and a chemical inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The endogenous expression of Her2 was altered by both overexpression and morpholino-knockdown approaches, and the results demonstrated that Her2 was both necessary and sufficient to promote the proliferation of neural progenitors by inhibiting the transcription of the cell cycle inhibitors cdkn1a, cdkn1ba, and cdkn1bb. Her2 knockdown caused premature neuronal differentiation, which indicates that Her2 is essential for inhibiting neuronal differentiation. At a later stage of neural development, Her2 could induce glial differentiation. The overexpression of Her2 constructs lacking the bHLH or WRPW domain phenocopied the effect of the morpholino knockdown, demonstrating the essential function of these two domains and further confirming the knockdown specificity. In conclusion, our data reveal that Her2 promotes progenitor proliferation and maintains progenitor characteristics by inhibiting neuronal differentiation. Together, these two mechanisms ensure the proper development of the neural progenitor cell pool.
    Document Type:
    Reference
    Product Catalog Number:
    17-10085
    Product Catalog Name:
    Magna ChIP™ A/G Chromatin Immunoprecipitation Kit - (Magna ChIP™ A/G Chromatin Immunoprecipitation Kit)