Search results for "Transfection"
showing 10 items of 581 documents
Toll-like receptor 3 mediates expression of clusterin/apolipoprotein J in vascular smooth muscle cells stimulated with RNA released from necrotic cel…
2010
Clusterin/Apolipoprotein J is a protein that is upregulated in a broad spectrum of diverse pathological processes. The predominant form is a secreted glycoprotein (sCLU) with cytoprotective and anti-inflammatory properties which shows enhanced expression in vascular smooth muscle cells (VSMC) following aortic injury and in atherosclerotic disease. Recent evidence indicates that during atherosclerosis, Toll-like receptors (TLRs) are activated in vascular cells by endogenous ligands. Here, we analyzed whether CLU expression in VSMC is controlled by TLRs, and stimulated by factors associated with or released by necrotic cells. Activation of TLR3 by the synthetic RNA analogue polyinosinic-polyc…
Nuclear receptors modulate the interaction of Sp1 and GC-rich DNA via ternary complex formation
2000
Binding sites for transcription factor Sp1have been implicated in the transcriptional regulation of several genes by hormones or vitamins, and here we show that a GC-rich element contributes to the retinoic acid response of the interleukin 1β promoter. To explain such observations, it has been proposed that nuclear receptors can interact with Sp1 bound to GC-rich DNA. However, evidence supporting this model has remained indirect. So far, nuclear receptors have not been detected in a complex with Sp1 and GC-rich DNA, and the expected ternary complexes in non-denaturing gels were not seen. In search for these missing links we found that nuclear receptors [retinoic acid receptor (RAR), thyroid…
Interaction of Neuronal Calcium Sensor-1 (NCS-1) with Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Affects Membrane Trafficki…
2001
Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated P…
Live cell imaging of duplex siRNA intracellular trafficking.
2015
Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging …
Differential regulation of apoptosis-associated genes by estrogen receptor alpha in human neuroblastoma cells
2012
Purpose: The neuroendocrinology of female sex hormones is of great interest for a variety of neuropsychiatric disorders. In fact, estrogens and estrogen receptors (ERs) exert neuromodulatory and neuroprotective functions. Here we investigated potential targets of the ER subtype alpha that may mediate neuroprotection and focused on direct modulators and downstream executors of apoptosis. Methods: We employed subclones of human neuroblastoma cells (SK-N-MC) stably transfected with one of the ER subtypes, ERalpha or ERbeta. Differences between the cell lines regarding the mRNA expression levels were examined by qPCR, changes on protein levels were examined by Western Blot and immunocytochemist…
Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery.
2007
In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to +45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN-DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human l…
Inulin-Ethylenediamine Coated SPIONs Magnetoplexes: A Promising Tool for Improving siRNA Delivery.
2015
An inulin based polycation (Inu-EDA) has been synthesized by the grafting of ethylenediamine molecules onto inulin backbone. The obtained inulin copolymer has been though to coat SPIONs (IC-SPIONs) and obtain stable magnetoplexes by complexation of IC-SPIONs with a model duplexed siRNA, for improving oligonucleotide transfection efficiency.The physical-chemical characteristics of IC-SPIONs and IC-SPIONs/siRNA magnetoplexes have been investigated by scanning and transmission electron microscopies, dynamic light scattering, FT-IR and qualitative surface elementary analysis. Cell compatibility and internalization in vitro of IC-SPIONs have been evaluated by MTS and fluorescence microscopy resp…
Proliferative activity and tumorigenic conversion: impact on cellular metabolism in 3-D culture
2001
Oxygen consumption, glucose, lactate, and ATP concentrations, as well as glucose and lactate turnover rates, have been studied in a three-dimensional carcinogenesis model of differently transformed rat embryo fibroblasts (spontaneously immortalized Rat1 and myc-transfected M1, and the ras-transfected, tumorigenic descendants Rat1-T1 and MR1) to determine metabolic alterations that accompany tumorigenic conversion. Various bioluminescence techniques, thymidine labeling, measurement of[Formula: see text] distributions with microelectrodes, and determination of cellular oxygen uptake rates (Q˙[Formula: see text]) have been applied. In the ras-transfected, tumorigenic spheroid types, the size d…
Corrigendum to "poly (ADP-ribose) polymerase inhibition synergizes with the NF-κB inhibitor DHMEQ to kill hepatocellular carcinoma cells" [Biochim. B…
2018
Fig. 1. The effects of the DHMEQ–Olaparib combination on HCC cells. (A) Cells were treated for 72 hwith the indicated concentrations of DHMEQ–Olaparib and cell viability was assessed by MTS assays. The DHMEQ–Olaparib combination showed synergistic inhibition of cell viability in Hep3B cells and additive inhibition in Huh7 cells. Combination index (CI) values are indicated above the bar. Data are expressed as percent cell growth and are the mean ± SD of three separate experiments (each of which was performed in triplicate). *p b 0.05 and **p b 0.01 versus each agent alone. (B) Cells were treated for 24 h with DHMEQ (μg/ml) or Olaparib (μM) alone or in combination, allowed to grow for 14 days…
Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis
1998
Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to t…