► A system established resulted in high expression level of an alpha7 type chimerical receptor. ► The receptor was purified in either an agonist bound form or an antagonist bound form. ► Purified ...receptor has been confirmed to be the intact mature receptor by mutiple methods.
Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT
3 chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT
3 receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.
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Although detailed cytogenetic analysis has been carried out in many types of cancer, there is little information on the chromosomal makeup of prostatic cancer cells. Karyological analyses of cell ...lines derived from both metastatic and primary prostatic carcinoma have been carried out by Q-, C-, and sequential banding techniques. The metastatic line, PC-3, isolated from a bone marrow specimen, is an established epithelial line which is tumorigenic in nude, athymic mice and forms colonies in semisolid agar suspension. A subline, PC-3/M, was isolated from a PC-3-induced mouse tumor. Karyotypic analysis of PC-3 by Q- and C-banding showed the cells to be aneuploid at all culture passage levels. The modal chromosome number shifted from 62 to 55 between the 5th and 50th passages. PC-3 has a unique karyotype. Chromosomes 2, 3, 5, 15, and Y were always absent. At least 11 different marker chromosomes were observed. The subline, PC-3/M, had a similar karyotype and retained the parental PC-3 markers. PC-3/M had a more restricted chromosomal frequency distribution range. Nearly 73% of the PC-3/M cells examined had 60 or 61 chromosomes in contrast to the wide distribution seen in PC-3. Silver staining for nucleolus organizer regions indicated that the number of functional nucleolus organizer regions in PC-3 was proportional to the number of acrocentric chromosomes. Banding analysis of PC-5-PI isolated from primary prostatic adenocarcinoma indicated that this line also had a characteristic karyotype with 28% pseudodiploid and 72% pseudotetraploid components. All metaphases examined were partially trisomic in chromosome 9 and lacked a demonstrable Y chromosome.
The clonal growth and serial propagation of rat esophageal epithelial cells in low serumcontaining medium has been achieved without feeder layers or conditioned medium. To date, a total of four lines ...have been developed and maintained for as many as 40 passages in culture. Growth of the cells was possible only after modifying the culture medium (PFMR-4) by reducing the calcium concentration from 1 to 0.1 mM, and by adding low levels of dialyzed fetal bovine serum and seven growth factors; i.e. epidermal growth factor, hydrocortisone, ethanolamine, phosphoethanolamine, insulin, transferrin, and cholera toxin. Cell lines have been developed from both explant outgrowths and enzyme dissociated esophagi. The epithelial nature of the cells was confirmed by electron microscopy and immunological methods. Clonal growth studies revealed that optimal cell growth occurred in medium containing 2.4% dialyzed fetal bovine serum and 0.1 mM calcium. Calcium levels of 0.3 mMor higher caused the cells to stratify and undergo terminal differentiation. Coating the culture dishes with collagen, or a combination of collagen, fibronectin, and bovine serum albumin, increased both the cell growth rate and the colony forming efficiency. The successful long term culture of rat esophageal epithelial cells permits their use as models in studies concerned with esophageal differentiation and carcinogenesis.
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4.
Ethical Communication Christians, Clifford G. & Merrill, John C
2013, 2009, May 2009
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Proponents of professional ethics recognize the importance of theory but also know that the field of ethics is best understood through real-world applications. This book introduces important ethical ...concepts through the lives of major thinkers ranging from Aristotle to Ayn Rand, John Stuart Mill to the Dalai Lama. Some two dozen contributors approach media ethics from five perspectives-altruistic, egoistic, autonomous, legalist, and communitarian-and use real people as examples to convey ethical concepts as something more than mere abstractions.
Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic ...property of ellagic acid, we tested its effects on the toxicity of benzoapyrene and benzoapyrene, 7,8-dihydrodiol and binding of benzoapyrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 micrograms/ml) enhanced the toxicity of benzoapyrene 7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of 1.5 and 3.0 micrograms/ml inhibited binding of benzoapyrene metabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzoapyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.
Using an explant/cell culture system, rat esophageal epithelial cells were transformed in vitro by exposure to N-nitroso-N-benzyl-N-methylamine (BMNA). Twelve esophageal explant cultures per group ...were exposed twice (at days 1 and 7) to 0.0, 2.5, 5.0 or 10.0 micrograms BMNA/ml of medium. After incubation for 60-90 days, epithelial cells in primary cultures treated with all three concentrations of BMNA could be subcultured and cell lines were developed. The number of primary cultures and the number of subsequently developed epithelial cell lines was carcinogen-dose-dependent. Cell lines could only be established from carcinogen treated explants. Electron microscopy revealed that the BMNA-treated cell lines contained morphological markers of esophageal epithelial cells; i.e., numerous tonofilaments and junctional complexes, even after prolonged subculture. By immunofluorescence, the cells reacted positively with antibodies prepared to mouse skin prekeratins (K1 and K2). Two cell lines (from the 5 micrograms BMNA/ml group) were able to grow in soft agar and produce palpable tumors upon injection into syngeneic recipients. These tumors possessed the histological features of squamous cell carcinomas.
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9.
Controversies in Media Ethics Gordon, A. David; Kittross, John Michael; Merrill, John C. ...
2011, 20120523, 2011-07-27, 2012-05-23
eBook
Controversies in Media Ethics offers students, instructors and professionals multiple perspectives on media ethics issues presenting vast "gray areas" and few, if any, easy answers. This third ...edition includes a wide range of subjects, and demonstrates a willingness to tackle the problems raised by new technologies, new media, new politics and new economics.
The core of the text is formed by 14 chapters, each of which deals with a particular problem or likelihood of ethical dilemma, presented as different points of view on the topic in question, as argued by two or more contributing authors. The 15th chapter is a collection of "mini-chapters," allowing students to discern first-hand how to deal with ethical problems. Contributing authors John A. Armstrong, Peter J. Gade, Julianne H. Newton, Kim Sheehan, and Jane B. Singer provide additional voices and perspectives on various topics under discussion.
This edition has been thoroughly updated to provide:
discussions of issues reflecting the breadth and depth of the media spectrum
numerous real-world examples
broad discussion of confidentiality and other timely topics
A Companion Website (www.routledge.com/textbooks/9780415963329) supplies resources for both students and instructors. You can also join the Controversies community on Facebook: http://www.facebook.com/CME3rd
Developed for use in media ethics courses, Controversies in Media Ethics provides up-to-date discussions and analysis of ethical situations across a variety of media, including issues dealing with the Internet and new media. It provides a unique consideration of ethical concerns, and serves as provocative reading for all media students.
Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride ...transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2 micrograms/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band of M(r) ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.
