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Source Author Title Publ. Year Bib. FN
Mbs/Embark 2004 Hamdy M. Embark Regulation of Renal Ion Channels by Serum and Glucocorticoid Inducible Kinase Isoforms, Ubiquitin Ligase Nedd4-2 and NHE3 Regulating Factor 2 in the Xenopus Laevis Oocyte Expression System 2004 no no


Fragments (Plagiarism, reviewed)

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Fragments (Plagiarism, unreviewed)

4 Fragments

[1.] Analyse:Mbs/Fragment 007 32 - Diskussion
Bearbeitet: 28. August 2017, 01:21 Hindemith
Erstellt: 28. August 2017, 01:21 (Hindemith)
Embark 2004, Fragment, KomplettPlagiat, Mbs, SMWFragment, Schutzlevel, ZuSichten

Typus
KomplettPlagiat
Bearbeiter
Hindemith
Gesichtet
No
Untersuchte Arbeit:
Seite: 7, Zeilen: 32-46
Quelle: Embark 2004
Seite(n): 30, Zeilen: 1ff
Xenopus oocytes as expression model for heterologous membrane proteins

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA [Gurdon et al., 1971].

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages [Dumont,1972]. In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system in which to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte [reviewed in Wagner et al., 2000].

1.8 Xenopus laevis oocytes and electrophysiological recording

One of the first and still most widely used assay system for quantifying an authentic protein biosynthetic process is the fully grown oocyte of the South African clawed frog, Xenopus laevis. The value of Xenopus laevis first became apparent in 1971, when Gurdon and co-workers discovered that the oocyte constitutes an efficient system for translating foreign messenger RNA (Gurdon et al., 1971).

The Xenopus oocyte is a cell specialized for the production and storage of proteins for later use during embryogenesis and developmentally divided into 6 stages (Dumont, 1972). In addition, the complex architecture of the frog oocyte includes the subcellular systems involved in the export and import of proteins. Therefore, the mRNA-microinjected oocyte is an appropriate system in which to study the synthesis of specific polypeptides, as well as the storage of particular proteins in various subcellular organelles and the export of others into the extracellular space. Moreover, the subcellular compartmentalization, as well as the structure and biochemical, physiological, and biological properties of the synthesized protein, may be examined from exogenous proteins in the injected oocyte (reviewed in Wagner et al., 2000).

Anmerkungen

The source is not mentioned here.

Sichter
(Hindemith)

[2.] Analyse:Mbs/Fragment 008 01 - Diskussion
Bearbeitet: 28. August 2017, 01:31 Hindemith
Erstellt: 28. August 2017, 01:28 (Hindemith)
Embark 2004, Fragment, KomplettPlagiat, Mbs, SMWFragment, Schutzlevel, ZuSichten

Typus
KomplettPlagiat
Bearbeiter
Hindemith
Gesichtet
No
Untersuchte Arbeit:
Seite: 8, Zeilen: 1ff (entire page)
Quelle: Embark 2004
Seite(n): 30, 31, 32, Zeilen: 30: 19ff; 31: 1ff; 32; 1ff
For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. While the nucleus resides in the animal pole [Nieuwkoop, 1977], more mRNA is present in the vegetable pole [Capco and Jeffery, 1982]. The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, [Dascal, 1987].

The primary advantage of using Xenopus oocytes for the expression of transporters is the ability to perform detailed electrophysiological recording using an in vivo system. In the simplest arrangement, the membrane is penetrated with a single microelectrode and the membrane potential is measured. The oocyte can easily be penetrated with two microelectrodes. This arrangement allows the use of one of the two classical methods: current clamp or voltage clamp. Most electrophysiological studies on oocytes were performed using the two-electrode voltage-clamp. The large size of the oocytes also permits extracellular recording of currents flowing through the cell membrane at various locations using a vibrating probe. The patch clamp method has been successfully applied in devitellinized oocytes for the study of single channels [Hamill et al., 1981].

Whole-cell voltage clamping of oocytes involves two electrodes inserted into the oocyte. The large size of the oocyte (about 1 mm in diameter and 0.5 to 1 μl in volume for stage V-VI oocytes) make this feasible, and is both the major advantage and disadvantage of the system. The advantage is that it is possible to insert multiple electrodes and injection needles into the same oocyte. Therefore, modulators of channel function can be injected inside the cell while recording, so that a rapid and direct response to an intracellular signal can be observed. The disadvantage is that the large size results in an extremely large membrane capacitance (about 150-200 nF), which causes a slow clamp setting time following voltage shifts. This makes it difficult to obtain any data during the first 1 to 2 msec of a hyper- or depolarization, the time during which rapidly activating voltage sensitive channels such as the cardiac sodium channel open. The large capacitance is not a serious problem in examining slow responses or ligand-gated responses in the absence of voltage shifts [Stuhmer, 1992]. Despite their advantages, several precautions should be taken into consideration.

Firstly, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high level can induce endogenous channels. [Tzounopoulos et al.,1995].

Secondly, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temprature & most experiments are carried out at room teperature. Hence, tempreture sensitive processes i.e. protein trafficking or kinethics may be altered.

Finally, since Xenopus oocytes may have different signaling pathways, precatuion should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA & PKC pathway. 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].

For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. While the nucleus resides in the animal pole (Nieuwkoop, 1977), more mRNA is present in the vegetable pole (Capco and Jeffery, 1982). The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl- conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, (Dascal, 1987).

The primary advantage of using Xenopus oocytes for the expression of transporters is the ability to perform detailed electrophysiological recording using an in vivo system. In the simplest arrangement, the membrane is penetrated with a single microelectrode and the membrane potential is measured. The oocyte can easily be penetrated with two microelectrodes. This

[page 31]

arrangement allows the use of one of the two classical methods: current clamp or voltage clamp. Most electrophysiological studies on oocytes were performed using the two-electrode voltage-clamp. The large size of the oocytes also permits extracellular recording of currents flowing through the cell membrane at various locations using a vibrating probe. The patch clamp method has been successfully applied in devitellinized oocytes for the study of single channels (Hamill et al., 1981).

Whole-cell voltage clamping of oocytes involves two electrodes inserted into the oocyte. The large size of the oocyte (about 1 mm in diameter and 0.5 to 1 μl in volume for stage V-VI oocytes) make this feasible, and is both the major advantage and disadvantage of the system. The advantage is that it is possible to insert multiple electrodes and injection needles into the same oocyte. Therefore, modulators of channel function can be injected inside the cell while recording, so that a rapid and direct response to an intracellular signal can be observed. The disadvantage is that the large size results in an extremely large membrane capacitance (about 150-200 nF), which causes a slow clamp setting time following voltage shifts. This makes it difficult to obtain any data during the first 1 to 2 msec of a hyper- or depolarization, the time during which rapidly activating voltage sensitive channels such as the cardiac sodium channel open. The large capacitance is not a serious problem in examining slow responses or ligand-gated responses in the absence of voltage shifts (Stuhmer, 1992).

Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (Tzounopoulos et al., 1995). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered (Wagner et al., 2000).

[page 32]

Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA and PKC pathway. 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).

Anmerkungen

The source is not mentioned here.

Sichter
(Hindemith)

[3.] Analyse:Mbs/Fragment 009 01 - Diskussion
Bearbeitet: 28. August 2017, 01:34 Hindemith
Erstellt: 28. August 2017, 01:34 (Hindemith)
Embark 2004, Fragment, KomplettPlagiat, Mbs, SMWFragment, Schutzlevel, ZuSichten

Typus
KomplettPlagiat
Bearbeiter
Hindemith
Gesichtet
No
Untersuchte Arbeit:
Seite: 9, Zeilen: 1-7
Quelle: Embark 2004
Seite(n): 32, Zeilen: 11ff
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. 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.
Anmerkungen

The source is not mentioned.

Sichter
(Hindemith)

[4.] Analyse:Mbs/Fragment 009 17 - Diskussion
Bearbeitet: 28. August 2017, 01:41 Hindemith
Erstellt: 28. August 2017, 01:41 (Hindemith)
Embark 2004, Fragment, KomplettPlagiat, Mbs, SMWFragment, Schutzlevel, ZuSichten

Typus
KomplettPlagiat
Bearbeiter
Hindemith
Gesichtet
No
Untersuchte Arbeit:
Seite: 9, Zeilen: figure 7
Quelle: Embark 2004
Seite(n): 51, Zeilen: figure 14
Mbs 009a diss

Fig. 7: The two-electrode voltage-clamp (adapted from Baumgartner et al., 1999). The voltage recording electrode eV monitors the membrane potential; this is compared with a command voltage Vc, and the magnified difference is applied to a current injection electrode, eI. A bath electrode eB serves as the return path for the injected current

Mbs 009a source

Fig. 14. The two-electrode voltage-clamp (adapted from Baumgartner et al., 1999). The voltage recording electrode eV monitors the membrane potential; this is compared with a command voltage Vc, and the magnified difference is applied to a current injection electrode, eI. A bath electrode eB serves as the return path for the injected current.

Anmerkungen

The source is not given here.

Sichter
(Hindemith)


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