Transfection cells

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor, (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors, (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and lOOnM. Dose-response curve for the effects seen in panel B. 

Several patients have exhibited a clinical picture resembling FHH, but no CaR mutations could be identified. These individuals also exhibited various forms of autoimmunity (e.g., antithyroid antibodies) and harbored anti-CaR antibodies that reduced the high CaQ+ -evoked stimulation of MAPK and PLC in cells transfected with the wild type receptor. Thus both antireceptor antibodies and mutations in the CaR can render the receptor resistant to activation by Caq+, producing a clinical picture of mild, PTH-dependent hypocalciuric hypercalcemia [3]. 

Lukacs, K.V., Lowrie, D.B., Stokes, R.W., Colston, M.J. (1993). Tumor cells transfected with a bacterial heat shock gene lose tumorigenicity and induce protection against tumors. J. Exp. Med. 178,343-348. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

A wide selection of monoclonal and polyclonal anti-Ca -ATPase antibodies have become available in recent years. Studies with these antibodies defined the localization of Ca " -ATPase in the sarcoplasmic reticulum of developing and mature skeletal muscles [60,262-270] and established a pattern of cross reactivity with various Ca -ATPase isoenzymes in the sarco(endo)plasmic reticulum [270-286] and in the plasma membrane [284,287-290] of skeletal, cardiac and smooth muscles. Antibodies have also proved useful in the quantitation of Ca -ATPase, both in muscles of diverse fiber types [291-294] and in COS-1 cells transfected with Ca -ATPase cDNA [97,103,126,127,129,215],... 

When assayed in HEK293 cells transfected with the cloned human, rat and guinea pig TRPVl, (23a) showed similar potencies. Not unexpeetedly, (23a) showed poor metabolic stability and a structure-activity study to optimize potency and drug-like properties was initiated. Modification on the left-handed A -aryl section showed that ... 

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

Besides this purported action on DAT, amphetamine has also been suggested to act upon the vesicular transporter as well. Pifl et al.87 examined COS cells transfected with cDNA for either DAT or the vesicular transporter, or both. A marked increase in DA release was noted in cells that expressed both DAT and the vesicular transporter when compared to the release from cells that express only DAT or the vesicular transporter. The mechanism of action for amphetamine was further defined with the work of Giros et al.59 In transgenic mice lacking the DAT, amphetamine did not produce hyperlocomotion or release DA. 

Nicotine increased DA levels both in vivo11,193 and in vitro. 94 196 Nicotine197 and its metabolites198 were found to both release and inhibit the reuptake of DA in rat brain slices, with uptake inhibition occurring at a lower concentration than that required for DA release. In addition, the (-) isomer was more potent than the (+) isomer.197 However, the effects of nicotine upon DA release and uptake were only apparent when brain slices were utilized because nicotine was unable to affect DA when a synaptosomal preparation was utilized.197 These results indicate that nicotine exerts its effects upon the DAT indirectly, most likely via nicotine acetylcholine receptors. This finding was supported by the results of Yamashita et al.199 in which the effect of nicotine on DA uptake was examined in PC 12 and COS cells transfected with rat DAT cDNA. Nicotine inhibited DA uptake in PC 12 cells that possess a nicotine acetylcholine receptor. This effect was blocked by the nicotinic antagonists hexamethonium and mecamylamine. Additionally, nicotine did not influence DA uptake in COS cells, which lack nicotinic acetylcholine receptors. 

Tang, F., K. Horie, and R. T. Borchardt. Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa , Pharm. Res. 2002, 29, 773-779... 

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

Parallel studies by Tallent et al. [65] have employed AtT-20 cells transfected with the fi receptor which couples to an endogenously expressed inwardly rectifying K+ channel. Prolonged application of DAMGO to these cells also desensitizes the ability of opiates to potentate the K+ current. The desensitization of the fi receptor in AtT-20 cells did not involve changes in the ability of the K+ channel to be activated since GTP analogs perfused into the opiate-treated cells increased K+ currents to a similar extent as in drug naive cells. 

The desensitization of the fi receptor was heterologous. In oocytes cotransfected with ft and serotonin receptors, chronic morphine treatment abolished morphine and serotonin potentiation of the K+ current [63]. Similarly, in AtT-20 cells transfected with the cloned fi receptor, chronic DAMGO treatment abolished the ability of opiates and somatostatin, acting via endogenous somatostatin receptors in these cells, to stimulate K+ conductance [65]. 

An example experiment is shown in Fig. 6.3A. Here, a dual probe RPA was performed on 1 /ig of total RNA purified from cells transfected with cap tail R-luc-4 sites and F-luc mRNA, with and without miCXCR4. The assay was performed on total RNA from untransfected cells to test for specificity. The level of R-luc-4 sites mRNA was not significantly different between cells transfected with miCXCR4 (+) and those not (—). 

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