Search results for "Transfection"

The selective β1-adrenoceptor antagonist nebivolol is a potential oestrogen receptor agonist with neuroprotective abilities

2010

Background and purpose:  Nebivolol, a selective β1-adrenoceptor antagonist mediating rapid vasodilating effects, is used clinically to treat hypertension. Recently, it was reported that nebivolol also acts as an oestrogen receptor (ER) agonist. To investigate the neuroprotective potential of oestrogens, we assessed the oestrogenic effects of nebivolol in several in vitro neuronal models. Experimental approach:  Human neuroepithelioma SK-N-MC cells stably transfected with human ER α and β, and mouse N2A neuroblastoma cells expressing human APP695SWE[N2Aswe, stably transfected with the Swedish mutation form of the Alzheimer-associated amyloid precursor protein (APPswe, K670M/N671L)] were incu…

The transport activity of the human cationic amino acid transporter hCAT-1 is downregulated by activation of protein kinase C

2001

1 The human cationic amino acid transporter hCAT-1 contains several consensus sequences for phosphorylation by protein kinase C (PKC). This study investigates the eAect of PKC activation on hCAT-1-mediated transport. 2 When expressed in Xenopus laevis oocytes, hCAT-1-mediated L-arginine transport was reduced to 44+3% after a 30 min treatment of the oocytes with 100 nM phorbol-12-myristate-13-acetate (PMA). 4a-phorbol-12,13-didecanoate (4a-PDD, 100 nM) had no eAect. 3 In EA.hy926 endothelial cells, maximal inhibition of hCAT-1-mediated L-arginine transport (to 3‐11% of control) was observed after treatment of the cells with 100 nM PMA for 4 h. A 20‐30 h exposure of the cells to 100 nM PMA le…

Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

2014

Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H(+)-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol,…

Mono- and diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7

1999

Polycyclic aromatic hydrocarbon (PAH)-type compounds induce at least two rat UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT1A7. Among the glucuronidation reactions of PAH metabolites studied, mono- and diglucuronide formation of benzo[a]pyrene and chrysene-3,6-diphenol showed the highest induction factors in rat liver microsomes. Availability of AHH-1 cells stably expressing UGT1A7 allowed us to study whether this PAH-inducible isoform could catalyze benzo[a]pyrene and chrysene-3,6-diphenol glucuronidation. It was found that UGT1A7 indeed catalyzed mono- and diglucuronide formation of both benzo[a]pyrene and chrysene 3,6-diphenols. V79 cell-expressed rat UGT1A6 also catalyzed these re…

Inhibiting proliferation in KB cancer cells by RNA interference-mediated knockdown of nicotinamide N-methyltransferase expression.

2011

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to asses…

Ganodermycin, a novel inhibitor of CXCL10 expression from Ganoderma applanatum

2011

CXCL10 (inducible protein-10) is a highly inducible chemoattractant, which contributes to the recruitment of inflammatory cells, such as macrophages and T-lymphocytes, and thereby has important roles in chronic inflammatory conditions. In a search for new inhibitors of CXCL10 expression in MonoMac6 cells, a novel compound, designated as Ganodermycin, was isolated from fermentations of the basidiomycete Ganoderma applanatum. The structure was determined by a combination of spectroscopic techniques. Ganodermycin inhibited the lipopolysaccharide (LPS)/interferon (IFN)-γ-induced CXCL10 promoter activity in transiently transfected MonoMac6 cells in a dose-dependent manner with IC(50) values of 1…

2015

Multidrug resistance is a prevailing phenomenon leading to chemotherapy treatment failure in cancer patients. In the current study two known cytotoxic pseudoguaianolide sesquiterpene lactones; neoambrosin (1) and damsin (2) that circumvent MDR were identified. The two cytotoxic compounds were isolated using column chromatography, characterized using 1D and 2D NMR, MS, and compared with literature values. The isolated compounds were investigated for their cytotoxic potential using resazurin assays and thereafter confirmed with immunoblotting and in silico studies. MDR cells overexpressing ABC transporters (P-glycoprotein, BCRP, ABCB5) did not confer cross-resistance toward (1) and (2), indic…

Addition of a signal peptide sequence to theα1D-adrenoceptor gene increases the density of receptors, as determined by [3H]-prazosin binding in the m…

2005

1 Both in mammalian tissues and in transfected cells, only low levels of α1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the α1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2 In the present study, we created gene constructs for six N-terminally truncated variants of the human α1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of α1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes…

Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and Rho GTPases in NIH 3T3 fibroblasts

2000

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of…

Correlations in palmitoylation and multiple phosphorylation of rat bradykinin B2 receptor in Chinese hamster ovary cells.

1999

Rat bradykinin B2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B2 receptor was isolated on oligo(dT)-cellulose using N-(epsilon-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA)30-mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isola…