von Dr. Sankarganesh Jeyaraj
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| [1.] Sj/Fragment 025 01 - Diskussion Zuletzt bearbeitet: 2016-11-23 19:54:55 WiseWoman | Embark 2004, Fragment, Gesichtet, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sj |
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| Untersuchte Arbeit: Seite: 25, Zeilen: 1-9 |
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| [However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation] can not be observed. Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (114). Exposing the Xenopus oocytes to the regulators of intracellular signaling such as PKC activator phorbol esters may unspecifically lead to internalization of the plasma membrane and the expressed proteins (115;116). In summary, the Xenopus oocyte system has the advantage that channels, receptors and transporters can rapidly be expressed and identified by their electrophysiological properties. Once cDNA clones have been isolated, oocytes are an excellent system for correlating structure with function using a combination of molecular biological and electrophysiological techniques and analyzed both biochemically and electro physiologically in an in vivo situation.
114. Waldegger,S, Raber,G, Sussbrich,H, Ruppersberg,JP, Fakler,B, Murer,H, Lang,F, Busch,AE: Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating IsK channels expressed in Xenopus oocytes. Kidney Int. 49:112-116, 1996 115. Vasilets,LA, Schwarz,W: Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases. J.Membr.Biol. 125:119-132, 1992 116. Loo,DD, Hirsch,JR, Sarkar,HK, Wright,EM: Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes. FEBS Lett. 392:250-254, 1996 |
However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation can not be observed (Wagner et al., 1996). Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (Waldegger et al., 1996). Exposing the Xenopus oocytes to the regulators of intracellular signaling such as PKC activator phorbol esters may unspecifically lead to internalization of the plasma membrane and the expressed proteins (Vasilets and Schwarz, 1992; Loo et al., 1996).
In summary, the Xenopus oocyte system has the advantage that channels, receptors and transporters can rapidly be expressed and analyzed both biochemically and electrophysiologically in an in vivo situation. The system can be used quite effectively as an assay for the functional cloning of channels that have only been identified by their electrophysiological properties. Once cDNA clones have been isolated, oocytes are an excellent system for correlating structure with function using a combination of molecular biological and electrophysiological techniques. |
The source is not given. |
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| [2.] Sj/Fragment 025 18 - Diskussion Zuletzt bearbeitet: 2016-11-23 19:42:01 WiseWoman | Embark 2004, Fragment, Gesichtet, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sj |
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| Untersuchte Arbeit: Seite: 25, Zeilen: 18-33 |
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| 3.3.1 In vitro RNA transcription
In-vitro cRNA transcription involves 2 consecutive steps i.e. linearization of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA. a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight. b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose. c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA. d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm. |
2.2.1 In vitro cRNA transcription
As illustrated in Fig. 12, in vitro cRNA transcription involves 2 consecutive steps i.e. linearisation of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA. a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight. b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose. c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA. d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm. |
The source is not mentioned. It is not surprising that identical procedures are used in different studies and it makes sense to describe those procedures with the same words, but it should be made transparent. |
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