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Angaben zur Quelle [Bearbeiten]

Autor     Nadeem Sheikh
Titel    Regulation of gene expression of hepcidin and of other proteins of the iron metabolism in the liver and in the extrahepatic tissues: in vivo and in vitro studies in different rat models.
Ort    Göttingen
Jahr    2006
Anmerkung    Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen
URL    http://d-nb.info/982460333/34

Literaturverz.   

no
Fußnoten    no
Fragmente    13


Fragmente der Quelle:
[1.] Iam/Fragment 006 20 - Diskussion
Zuletzt bearbeitet: 2014-03-12 19:44:25 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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Interleukin (IL)-6 itself from the group of IL-6-type cytokines and IL-1β together with tumor necrosis factor (TNF)-α from the IL-1-type cytokine group are considered to be the major mediators of the acute-inflamamtion. At the inflammatory sites, IL-6 is produced by macrophages, endothelial cells and fibroblasts (Ramadori and Christ 1999). The release of mature IL-1β by macrophages seems to take place only during or after cell death (Perregaux and Gabel 1998). TNF-α is synthesized mainly by mononuclear phagocytes recruited at the sites of damage and by tissue macrophages. While IL-6, IL-1β, and TNF-α are the inducers of acute phase protein gene expression, other cytokines were shown to modulate this expression (Ramadori and Christ 1999).

Perregaux DG, Gabel CA (1998) Post-translational processing of murine IL-1: evidence that ATP-induced release of IL-1 alpha and IL-1 beta occurs via a similar mechanism. J Immunol 160:2469-2477

Ramadori G, Christ B (1999) Cytokines and the hepatic acute-phase response. Semin Liver Dis 19:141-155

Interleukin- (IL) 6 from the group of IL-6-type cytokines and IL-1β together with tumor necrosis factor (TNF)-α from the group of IL-1-type cytokine are considered to be the major mediators of the APR. At the inflammatory sites, IL-6 is produced by macrophages, endothelial cells, and fibroblasts (Ramadori and Christ, 1999). The release of mature IL-1β by macrophages seems to take place only during or after cell death (Perregaux and Gabel, 1998). TNF-α is synthesized mainly by mononuclear phagocytes recruited at the sites of damage and by tissue macrophages (Ramadori and Christ, 1999). While IL-6, IL-1β, and TNF-α are the inducers of acute-phase protein gene expression, other cytokines (Table 1) were shown to modulate this expression (Moshage, 1997).

Moshage H. Cytokines and the hepatic acute phase response. J Pathol 181: 257-266, 1997.

Perregaux DG and Gabel CA. Post-translational processing of murine IL-1: evidence that ATP-induced release of IL-1{alpha} and IL-1{beta} occurs via a similar mechanism. J Immunol 160: 2469-2477, 1998.

Ramadori G and Christ B. Cytokines and the hepatic acute-phase response. Semin Liver Dis 19: 141-155, 1999.

Anmerkungen

Nearly identical - nevertheless nothing has been marked as a citation.

Sichter
(Graf Isolan) Schumann

[2.] Iam/Fragment 011 22 - Diskussion
Zuletzt bearbeitet: 2014-03-12 19:44:33 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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Once it became obvious that the liver is injured after irradiation, the individual liver cell types would be introduced in culture to investigate a hierarchy of the events triggering after irradiation in the liver. Besides the ability to respond to the cytokine action, liver parenchyma and non-parenchaymal cells express a great variety of receptors for chemokines, cytokines, growth factors, and prostaglandins and represent therefore the major target for a multiple set of mediators involved in defense reaction after liver irradiation (Christiansen et al. 2007).

Christiansen H, Sheikh N, Saile B, Reuter F, Rave-Frank M, Hermann RM, Dudas J, Hille A, Hess CF, Ramadori G (2007) x-Irradiation in rat liver: consequent upregulation of hepcidin and downregulation of hemojuvelin and ferroportin-1 gene expression. Radiology 242:189-197

Once it became obvious that the liver is a primary target organ for the APR, the individual liver cell types were introduced in culture to investigate a hierarchy of the events triggering the full APR in the liver. Besides the ability to respond to the cytokine action, different cell types within the liver are also able to express IL-1β, TNF-α, IL-6, and other modulator cytokines of the hepatic APR (Ramadori and Christ, 1999). [...] Hepatocytes express a great variety of receptors for cytokines, growth factors, and prostaglandins and therefore, represent the major target for a multiple set of mediators involved in both systemic and local host defense reactions.

Ramadori G and Christ B. Cytokines and the hepatic acute-phase response. Semin Liver Dis 19: 141-155, 1999.

Anmerkungen

Adapted to fit Iam's subject, but still in large parts identical. Nothing has been marked as a citation.

Sichter
(Graf Isolan) Schumann

[3.] Iam/Fragment 015 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:03:26 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

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2. MATERIALS

2.1 Animals

Male Wistar rats (about 200 g body weight) were purchased from Harlan-Winkelmann (Borchen, Germany) and kept under standard conditions with 12-hours light/dark cycles and access to fresh water and food pellets ad libitum at room temperature of 19-23°C. The rats consumed 12-15 g food (rat diet "ssniff", Spezialitäten GmbH, Soest, Germany) and 12-25 ml water per day and had a 30-40 g gain of weight per week. Animals were used for the experiments not earlier than 6 days after arrival. The preparation of hepatocytes was performed during the first 3 hours of the light phase.

2. MATERIALS

2.1 Animals

Male Wistar rats (about 200 g body weight) were purchased from Harlan-Winkelmann (Borchen, Germany) and kept under standard conditions with 12-hours light/dark cycles, access to fresh water and food pellets ad libitum at room temperature of 19-23°C. The rats consumed 12-15 g food (rat diet "ssniff", Spezialitäten GmbH, Soest, Germany) and 12-25 ml water per day and had a 30-40 g gain of weight per week. Animals were used for the experiments not earlier than 6 days after arrival. The preparation of hepatocytes was performed during the first 3 h of the light phase.

Anmerkungen

Identical; not marked as a citation.

Sichter
(Graf Isolan) Schumann

[4.] Iam/Fragment 025 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:29 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

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3.1.2 Isolation of rat hepatocytes and irradiation

Hepatocytes were isolated from male Wistar rats by circulating perfusion with collagenase essentially as described previously (Seglen 1972).

3.1.2.a. Liver perfusion

After laparotomy, the vena portae was canulated, vena cava inferior was ligated above the diaphragm to prevent flow of the perfusion media into a whole body circulation. Finally, the vena cava inferior was cut beneath the liver and canulated. The liver was perfused in non-recirculative mode through the portal vein with 150-200 ml CO2-enriched preperfusion medium at a flow rate of 30 ml/min until the liver was free from blood. To break down components of extracellular matrix, the liver was perfused in recirculative mode with collagenase perfusion medium until it started to feel soft (about 7-11 min).


Seglen PO (1972) Preparation of rat liver cells. I. Effect of Ca 2+ on enzymatic dispersion of isolated, perfused liver. Exp Cell Res 74:450-454

3.1.1 Isolation of rat hepatocytes

Hepatocytes were isolated from male Wistar rats by circulating perfusion with collagenase essentially as described previously (Seglen, 1973; Katz et al., 1979).

3.1.1.I Liver perfusion

After laparotomy, the vena portae was canulated, vena cava inferior was ligated above the diaphragm to prevent flow of the perfusion media into a whole body circulation. Finally, the vena cava inferior was cut beneath the liver and canulated. The liver was perfused in non-recirculative mode through the portal vein with 150-200 ml CO2-enriched preperfusion medium at a flow rate of 30 ml/min until the liver was free from blood. To break down components of extracellular matrix, the liver was perfused in recirculative mode with collagenase perfusion medium until it started to feel soft (about 7-11 min).

Anmerkungen

Though identical nothing has been marked as a citation.

Sichter
(Graf Isolan) Schumann

[5.] Iam/Fragment 026 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:24 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

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3.1.2.b. Preparation of the hepatocyte suspension

After perfusion, the liver was excised and transferred into a sterile glass beaker filled with culture medium M 199 with additives. Glisson’s capsule, i. e. collagen tissue around the liver, was carefully removed and discarded. To obtain a cell suspension, the tissue was disrupted mechanically using sterile forceps. Connective tissue and remainder of the liver capsule as well as big cell aggregates were removed by filtration of the primary cell suspension through a nylon mesh (pore-size 79 μm). Non-parenchymal cells and cell debris were removed by numerous selective sedimentations (20 g, 2 min, 4°C) in wash medium. After the last centrifugation, hepatocytes were suspended in medium M 199 with additives. 50 ml of M 199 was added per 1 g of wet weight of the sedimented cells; the cell suspension typically had a density of about 106/2.5 ml.

3.1.2.c. Media and solutions for hepatocyte preparation and culture

All media and solutions for cell culture were prepared in double distilled water, further purified by sterile filtration and stored at 4°C. All solutions were prepared not more than one day before the isolation.

Krebs-Ringer stock solution

    Final concentration
NaCl   120 mM
KCl   4.8 mM
MgSO4×7H2O   1.2 mM
KH2PO4   1.2 mM
NaHCO3   24.4 mM

The solution was equilibrated with carbogen and pH was adjusted to 7.35 [sic]

Pre-perfusion medium (prepared in 1X Krebs-Ringer solution)

    Final concentration
EGTA   0.25 mM

Collagenase perfusion medium (prepared in 1X Krebs-Ringer solution)

    Final concentration
HEPES   15 mM
CaCl2×2H2O   4 mM
[Collagenase 50 mg

The medium was prepared directly prior to isolation, equilibrated with carbogen for 30 min and finally sterile filtered.]

[Page 25]

3.1.1.II Preparation of the hepatocyte suspension

After perfusion, the liver was excised and transferred into a sterile glass beaker filled with culture medium M 199 with additives. Glisson’s capsule, i.e. collagen tissue around the liver, was carefully removed and discarded. To obtain a cell suspension, the tissue was disrupted mechanically using sterile forceps. Connective tissue and remainder of the liver capsule as well as big cell aggregates were removed by filtration of the primary cell suspension through a nylon mesh (pore-size 79 μm). Non-parenchymal cells and cell debris were removed by numerous selective sedimentations (20 g, 2 min, and 4°C) in wash medium. After the last centrifugation, hepatocytes were suspended in medium M 199 with additives. 50 ml of M 199 was added per 1 g of wet weight of the sedimented cells; the cell suspension typically had a density of about 106/2.5 ml.

[Page 26]

3.1.1.III Media and solutions for hepatocyte preparation and culture

All media and solutions for cell culture were prepared in double distilled water, further purified by sterile filtration and stored at 4°C. All solutions were prepared not more than one day before the isolation.

Krebs-Ringer stock solution
  For 1l Final concentration
NaCl 7 g 120 mM
KCl 0.36 g 4.8 mM
MgSO4×7H2O 0.296 g 1.2 mM
KH2PO4 0.163 g 1.2 mM
NaHCO3 2.016 g 24.4 mM
dd H2O to 1 l  
The solution was equilibrated with carbogen and pH was adjusted to 7.35 [sic]
Pre-perfusion medium
  For 1 l Final concentration
EGTA 95.1 mg 0.25 mM
Krebs-Ringer stock solution to 1 l  
Collagenase perfusion medium
  For 100 ml Final concentration
HEPES 360 mg 15 mM
CaCl2×2H2O 58.8 mg 4 mM
Collagenase 50 mg
Krebs-Ringer stock solution to 100 ml  
The medium was prepared directly prior to isolation, equilibrated with carbogen for 30 min and finally sterile filtered.
Anmerkungen

Though identical nothing has been marked as a citation.

Typical C&P-mistake: The author has overlooked that the "6" in "106/2.5 ml" is an exponent and not just another digit.

Sichter
(Graf Isolan) Schumann

[6.] Iam/Fragment 027 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:19 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

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Collagenase 50 mg
The medium was prepared directly prior to isolation, equilibrated with carbogen for 30 min and finally sterile filtered.


Wash medium

    Final concentration
HEPES/NaOH pH 7.4   20 mM
NaCl   120 mM
KCl   4.8 mM
MgSO4×7H2O   1.2 mM
KH2PO4   1.2 mM
Bovine serum albumin   0.4%

Medium M 199 with additives

M199 with Earle’s salts without NaHCO3   Final concentration
Glucose×H2O   5.5 mM
HEPES   15 mM
NaHCO3   18 mM
Bovine serum albumin   0.4%
The medium was equilibrated with carbogen until pH reached a value of 7.35. Finally, the medium was sterile filtered.
[page 26]

Collagenase 50 mg

[...]

The medium was prepared directly prior to isolation, equilibrated with carbogen for 30 min and finally sterile filtered.

[page 27]

Wash medium
  For 1l Final concentration
HEPES/NaOH pH 7.4 4.77 g 20 mM
NaCL 7.00 g 120 mM
KCl 0.36 g 4.8 mM
MgSO4×7H2O 0.30 g 1.2 mM
KH2PO4 0.16 g 1.2 mM
Bovine serum albumin 4.00 g 0.4%
dd H2O to 1 l  
Medium M 199 with additives
  For 1l Final concentration
M199 with Earle’s salts without NaHCO3 1 l
Glucose × H2O 1.1 g 5.5 mM
HEPES 3.6 g 15 mM
NaHCO3 1.5 g 18 mM
Bovine serum albumin 4.0 g 0.4%
The medium was equilibrated with carbogen until pH reached a value of 7.35. Finally, the medium was sterile filtered.
Anmerkungen

Continuation of Fragment 026 01.

Sichter
(Singulus) Schumann

[7.] Iam/Fragment 036 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:03:32 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

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3.2.2.b. Thermal cycler amplification program

The amplification was performed at 50 °C for 2 min, 95°C for 2 min., 95°C for 15 seconds to 60°C for 30 seconds for 45 cycles (Figure 8) in an ABI prism 7000 sequence detection system. All samples were assayed in duplicate. Expression of different genes was analyzed using Platinum SYBR Green qPCR mix UDG. The PCR amplification program was followed by dissociation curve protocol for controlling the specificity of the PCR products. Specific temperature of dissociation of the PCR product was calculated by the Primer Express software. Curves of amplification were analyzed to measure the Ct value in the linear range of the amplification. The results were normalized to the house keeping gene and fold change expression was calculated using Ct values by Prism Graph Pad 4 software.

36a diss Iam.png

Figure 8: Thermal cycler amplification program for the quantitative real-time PCR amplification of the mRNA using Platinum® SYBR® Green qPCR SuperMix UDG, specific forward, reverse primer and template cDNA in ABI Prism® 7000 Sequence Detection System by Applied Biosystems. Stage 1; 2 minutes incubation at 50°C, Stage 2; 2 minutes incubation at 95°C for hot start, Stage 3; 15 sec at 95°c and 30sec at 60°C for 45 repeats, Stage 4; 15 sec 95°C, 15sec 60°C and 15 sec 95°C to get dissociation curve.

[Page 30]

3.2.1.II Thermal cycler amplification program

The amplification was performed at 50 °C for 2 min., 95°C for 2 min., 95°C for 15 sec to 60°C for 30 sec for 45 cycles (Figure 2) in an ABI prism 7000 sequence detection system. All samples were assayed in duplicate. Expression of different genes was analysed using Platinum SYBR Green qPCR mix UDG. The PCR amplification program was followed by dissociation curve protocol for controlling the specificity of the PCR products. Specific temperature of dissociation of the PCR product was calculated by the Primer Express software. Curves of amplification were analysed to measure the Ct value in the linear range of the amplification. The results were normalized to the

[Page 31]

housekeeping gene and fold change expression was calculated using Ct values by Prism Graph Pad 4 software.

36a source Iam.png

Figure 3: Thermal cycler amplification program for the quantitative real-time PCR amplification of the mRNA using Platinum® SYBR® Green qPCR SuperMix UDG, specific forward, reverse primer pairs and template cDNA in ABI Prism® 7000 Sequence Detection System by Applied Biosystems. Stage 1; 2 min. incubation at 50°C, Stage 2; 2 min. incubation at 95°C for hot start, Stage 3; 15 sec at 95°C and 30 sec at 60°C for 45 repeats. Stage 4; 15 sec at 95°C; 15 sec at 60°C and 15 sec at 95°C to get dissociation curve.

Anmerkungen

But for some "corrections" identical up to the breaks between words ("50 °C" vs. "95°C" in both texts), though nothing has been marked as a citation. Iam's "figure 8" is also identical to Sheiks "figure 3"

Sichter
(Graf Isolan) Schumann

[8.] Iam/Fragment 037 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:03:37 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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3.2.2.c. Primer designing

Primers for different genes were designed using the program “Primer Express” (ABI System) and the gene bank data (http://www.ncbi.nlm.nih.gov). All the primer sets used for real-time PCR are listed in the Table 1.

3.2.3 Isolation of total RNA

3.2.3.a. RNA isolation procedure using silica columns

The isolation of total RNA from cultured rat liver hepatocytes, Kupffer cells, myofibroblasts and laser-microdissected samples of the liver was conducted using the NucleoSpin® RNAII kit (Macherey-Nagel) in accordance to the protocol for cultured animal cells.

3.2.3.b. Isolation of RNA by density-gradient ultracentrifugation

Total RNA was isolated from the liver by means of guanidine isothiocyanate extraction, cesium chloride density-gradient ultracentrifugation and ethanol precipitation according to method of Chirgwin (Chirgwin et al. 1979). This method is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high.

Cell lysis: The cells were rapidly lysed in guanidine isothiocyanate-containing buffer, which ensures inactivation of RNases. The lysates were layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA settle down at the bottom of the tubes as a pallet [sic]. The RNA was recovered by dissolving the pellet. The recovery of RNA was usually excellent if the capacity of the gradient did not exceed.

Homogenization of the tissue sample: About 100 mg of frozen tissue was homogenized with Ultra-Turrax TP 18/10 homogenizer 3 times for 10 sec each in 3 ml of ice-cold GITC buffer with freshly added Antifoam A (Sigma). The homogenates were centrifuged for 10 min at 3,500 rpm in a Rotixa /RP centrifuge (Hettich) at 4°C to pellet connective tissue and large cell debris.

CsCl gradient and ultra centrifugation: To prepare the gradient 2 ml of CsCl buffer was poured into 5-ml polyallomer ultracentrifuge tubes (6 per preparation). The cleared guanidine lysed samples were carefully layered on top of the CsCl buffer.


Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299

[Page 32]

3.2.1.IV Primers designing

Primers for different genes were designed using the program “Primer Express” (ABI System) and the gene bank data (http://www.ncbi.nlm.nih.gov). All the primer sets used for real-time PCR are listed in the Table 1.

3.2.2 Isolation of total RNA

3.2.2.I RNA isolation procedure using silica columns

The isolation of total RNA from cultured rat hepatocytes was conducted using the NucleoSpin® RNAII kit (Macherey-Nagel) in accordance to the protocol for cultured animal cells.

[Page 34]

3.2.2.IV Isolation of RNA by density-gradient ultracentrifugation

Total RNA was isolated from the liver, the skeletal muscle and extrahepatic organs by means of guanidine isothiocyanate extraction, cesium chloride density-gradient ultracentrifugation and ethanol precipitation according to method of Chirgwin (Chirgwin et al, 1979). This method is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high.

Cell lysis

The cells were rapidly lysed in guanidine isothiocyanate-containing buffer, which ensures inactivation of RNases. The lysates were layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA settle down at the bottom of the tubes as a pellet. The RNA was recovered by dissolving the pellet. The recovery of RNA was usually excellent if the capacity of the gradient does not exceed.

Homogenization of the tissue sample

About 100 mg of frozen tissue was homogenized with ultra-turrax TP 18/10 homogenizer 3 times for 10 sec each in 3 ml of ice-cold GITC buffer with freshly added antifoam A (Sigma). The homogenates were centrifuged for 10 min at 3,500 rpm in a Rotixa/RP centrifuge (Hettich) at 4°C to pellet connective tissue and large cell debris.

[Page 35]

CsCl gradient and ultra centrifugation

To prepare the gradient 2 ml of CsCl buffer was poured into 5-ml polyallomer ultracentrifuge tubes (6 per preparation). The cleared guanidine lysed samples were carefully layered on top of the CsCl buffer.


Chirgwin JM, Przybyla AE, MacDonald RJ and Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294- 5299, 1979.

Anmerkungen

Slightly adapted, but otherwise nearly identical, though not marked as a citation.

Sichter
(Graf Isolan) Schumann

[9.] Iam/Fragment 038 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:03:42 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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Untersuchte Arbeit:
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Seite(n): 35, 36, Zeilen: 35:4-25; 36:1ff
[The samples were centrifuged] overnight (21 h) at 35,000 rpm in a Kontron TST55 rotor at 20°C. The supernatants were carefully removed by aspiration and the transparent gelatin-like RNA pellets were gently washed (preserving undisturbed) with 200 μl of 70% ethanol at room temperature. The pellets were reconstituted in 200 μl of RNase-free water by pipetting and transferred into sterile 1.5 ml eppendorf tubes and the procedure was immediately continued to RNA precipitation.

RNA precipitation: The RNA was precipitated with 450 μl of 100% ethanol in the presence of sodium acetate, pH 5.4 (20 μl of 2 M solution per pellet) overnight at –20°C. The RNA precipitates were centrifuged for 30 min at 12,000 rpm in an Eppendorf bench-top centrifuge at 4°C to get RNA pellet.

Washing of the RNA pellet: Supernatants were discarded and pellet was washed with 200 μl of ice-cold 70% ethanol to remove all traces of sodium acetate. The RNA precipitates were centrifuged as described above, the supernatants were discarded and the pellet was dried for 30 min. at room temperature.

Reconstitution of RNA: The pellets were reconstituted in 100 μl of RNase-free water. To determine the concentration and purity of the RNA obtained, the aliquot of RNA sample was diluted 1:100 in RNase-free H2O and the concentration was measured at 260 nm and 280 nm by spectrophotometer (GeneQuant II, Pharmacia Biotech).

Solutions used for Ultracentrifugation

Guanidine isothiocyanate (GITC) buffer

    Final concentration
Guanidine isothiocyanate   4 M
0.25 M sodium citrate   25 mM
N-lauroylsarcosyl   0.5%

The solution was sterile filtered and stored in the dark at 4°C. β-Mercaptoethanol was added just prior to use at a ration of 1 to 100 μl of GITC buffer.

Cesium chloride (CsCl) buffer

    Final concentration
Cesium chloride   5.7 M
0.25 M sodium citrate   25 mM
0.5 M EDTA   100 mM

pH was adjusted with 0.25 M citric acid to 7.5; the solution was dissolved in RNase-free [H2O, sterile filtered and stored at room temperature.]

[Page 35]

The samples were centrifuged overnight (21 h) at 35,000 rpm in a Kontron TST55 rotor at 20°C. The supernatants were carefully removed by aspiration and the transparent gelatin-like RNA pellets were gently washed (preserving undisturbed) with 200 μl of 70% ethanol at room temperature. The pellets were reconstituted in 200 μl of RNase-free water by pipetting and transferred into sterile 1.5 ml eppendorf tubes and the procedure was immediately continued to RNA precipitation.

RNA precipitation

The RNA was precipitated with 450 μl of 100% ethanol in the presence of sodium acetate, pH 5.4 (20 μl of 2 M solution per pellet) overnight at –20°C. The RNA precipitates were centrifuged for 30 min at 12,000 rpm in an Eppendorf bench-top centrifuge at 4°C to get RNA pellet.

Washing of the RNA pellet

Supernatants were discarded and pellets were washed with 200 μl of ice-cold 70% ethanol to remove all traces of sodium acetate. The RNA precipitates were centrifuged as described above, the supernatants were discarded and the pellets were dried for 30 minutes at room temperature.

Reconstitution of RNA

The pellets were reconstituted in 100 μl of RNase-free water. To determine the concentration and purity of the RNA obtained, the aliquot of RNA sample was diluted 1:100 in RNase-free H2O and the concentration was measured at 260 nm and 280 nm by spectrophotometer (GeneQuant II, Pharmacia Biotech).

[Page 36]

Solutions used for Ultracentrifugation

Guanidine isothiocyanate (GITC) buffer
  For 200 ml Final concentration
Guanidine isothiocyanate 94.53 g 4 M
0.25 M sodium citrate 20 ml 25 mM
N-lauroylsarcosyl 1 g 0.5%
RNase-free H2O to 200 ml  

The solution was sterile filtered and stored in the dark at 4°C. β-Mercaptoethanol was added just prior to use at a ration of 1 to 100 μl of GITC buffer.

Cesium chloride (CsCl) buffer
  For 200 ml Final concentration
Cesium chloride 192 g 5.7 M
0.25 M sodium citrate 20 ml 25 mM
0.5 M EDTA 40 ml 100 mM
RNase-free H2O to 200 ml  

pH was adjusted with 0.25 M citric acid to 7.5; the solution was sterile filtered and stored at room temperature.

Anmerkungen

Nearly identical, nevertheless not marked as a citation.

Sichter
(Graf Isolan) Schumann

[10.] Iam/Fragment 039 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:56 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

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[pH was adjusted with 0.25 M citric acid to 7.5; the solution was dissolved in RNase-free] H2O, sterile filtered and stored at room temperature.

3.2.4 Northern blot analysis

Northern blot analysis is a method to quantify RNA expression. The RNA is separated in a denaturing formaldehyde/agarose gel, transferred by capillary transfer to a nylon membrane and fixed by UV cross linking. The RNA of interest is identified by hybridization with a specific radiolabeled cDNA probe. All solutions used for the northern blot were autoclaved, the electrophoresis and blot chambers, gel plates and combs were kept in freshly prepared 0.1% DEPC solution for 10-20 min before use to inactivate RNases.

3.2.4.a. Preparation of RNA samples

Each RNA probe (5-10 μg of total RNA) in a volume not more than 5 μl was mixed with 7.5 μl of sample buffer. In case of dilute sample where the volume of the probe exceeded 5 μl, the sample was concentrated in a vacuum centrifuge until the volume was reduced to 5 μl. RNA probes mixed with sample buffer were denatured by heating at 65°C for 10 min. Afterwards, the samples were briefly cooled on ice and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge. Each sample was mixed with 3 μl of loading buffer and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge.

3.2.4.b. Electrophoresis

The electrophoresis was performed at constant voltage of 80 V for 1-1.5 h. After electrophoresis, the quality of RNA was estimated under UV transilluminator built-in Eagle Eye™ system (Stratagene); the gel was photographed and the procedure was immediately continued to blotting.

3.2.4.c. Transfer of RNA to nylon membrane

After the separation of RNA in the gel, it was transferred to a nylon membrane by capillary transfer method. A plastic tray was filled with 500 ml of 20X SSC and a piece of Whatman 3MM filter was soaked in 20X SSC and swaddled over the glass plate placed over the tray with both ends hanging into buffer to act as wick. The transfer was carried out overnight. After the transfer, RNA was fixed on the membrane by UV cross linking for 2 min from both sides using Stratalinker™ 180 (Stratagene) set at “autocross link” mode.

Pre-hybridization: Afterwards, the membrane was placed into a hybridization tube and any bubbles between the membrane and internal wall of the tube were carefully removed.

[Page 36]

pH was adjusted with 0.25 M citric acid to 7.5; the solution was sterile filtered and stored at room temperature.

3.2.3 Northern blot analysis

Northern blot analysis is a method to quantify RNA expression. The RNA is separated in a denaturing formaldehyde/agarose gel, transferred by capillary transfer to a nylon membrane and fixed by UV cross linking. The RNA of interest is identified by hybridization with a specific radiolabeled cDNA probe. All solutions used for the northern blot were autoclaved, the electrophoresis and blot chambers, gel plates and combs were kept in freshly prepared 0.1% DEPC solution for 10-20 min before use to inactivate RNases.

3.2.3.I Preparation of RNA samples

Each RNA probe (5-10 μg of total RNA) in a volume not more than 5 μl was mixed with 7.5 μl of sample buffer. In case of dilute sample where the volume of the

[Page 37]

probe exceeded 5 μl, the sample was concentrated in a vacuum centrifuge until the volume was reduced to 5 μl. RNA probes mixed with sample buffer were denatured by heating at 65°C for 10 min. Afterwards, the samples were briefly cooled on ice and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge. Each sample was mixed with 3 μl of loading buffer and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge.

[...]

3.2.3.III Electrophoresis

The electrophoresis was performed at constant voltage of 80 V for 1-1.5 h. After electrophoresis, the quality of RNA was estimated under UV transilluminator built-in Eagle Eye™ system (Stratagene); the gel was photographed, and the procedure was immediately continued to blotting.

3.2.3.IV Transfer of RNA to nylon membrane

After the separation of RNA in the gel, it was transferred to a nylon membrane by capillary transfer method. The system for transfer was assembled as depicted in Figure 4. A plastic tray was filled with 500 ml of 20×SSC. A piece of Whatman 3MM filter was soaked in 20×SSC and swaddled over the glass plate placed over the tray with both ends hanging into buffer to act as wick. [Any bubbles between the filter and glass plate were smoothed out. The gel was carefully placed upside down over the filter. The bubbles between the gel and wick were carefully removed. The nylon membrane wetted in 2×SSC was placed on top of the gel and smoothed. Two other pieces of Whatman 3MM filters soaked in 2×SSC were sequentially flat laid on top of nylon filter and smoothed. A stack]

[Page 38]

[of paper towels was placed on top, covered with another glass plate and pressed with 1 kg blotting weight.] The transfer was carried out overnight. After the transfer, RNA was fixed on the membrane by UV cross linking for 2 min from both sides using Stratalinker™ 180 (Stratagene) set at “autocross link” mode.

[...]

Pre-hybridization

Afterwards, the membrane was placed into a hybridization tube, and any bubbles between the membrane and internal wall of the tube were carefully removed.

Anmerkungen

Identical, without a hint that Iam is not the author of this text.

In "3.2.4.c." Iam has left out some of the original text, thus in fact the description of the procedure is incomplete.

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(Graf Isolan) Schumann

[11.] Iam/Fragment 046 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:39 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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3.3.3 Enzyme-Linked Immunosorbent Assay (ELISA)

To measure MIP-2/CXCL2 concentration in rat serum, the Quantikine® M rat CXCL2 immunoassay kit (R&D Systems, Wiesbaden, Germany), based on solid phase ELISA, was used.

3.3.3.a. Reagent preparation

Since all samples should be pipette within 15 min, reagents needed for the assay were prepared prior to assay procedure. All reagents were provided with Quantikine® immunoassay kit.

3.3.3.b. Rat CXCL2 control

The control provided with kit, was reconstituted with 1 ml double distilled water.

3.3.3.c. Rat CXCL2 conjugate concentrate

For 96 wells

Conjugate concentrate 0.5 ml

Conjugate diluent 11 ml

3.3.3.d. Washing buffer

For 96 wells

Washing buffer concentrate 25 ml

ddH2O to 625 ml

3.3.3.e. Substrate solution

Equal volumes of color reagents A and B, provided by kit, were mixed together, and solution was used with 15 min.

3.3.3.f. Standard and sample preparation

The standards were reconstituted with 2 ml of calibrator diluent. This stock solution (2000 pg/ml) was used to prepare a serial dilution ranging from 31.2 pg/ml to 2000 pg/ml. Calibrator diluent served as zero standard. Prior to assay, serum samples were diluted 2 fold into calibration diluent.

3.3.1 Enzyme-Linked Immunosorbent Assay (ELISA)

To measure IL-6, IL-1β, TNF-α and IFN-γ concentration in rat serum, the Quantikine® rat IL-6, IL-1β, TNF-α and IFN γ immunoassay kit (R&D Systems, Wiesbaden, Germany), based on solid phase ELISA, was used.

[page 50]

3.3.1.II Reagent preparation

Since all samples should be pipette within 15 min, reagents needed for the assay were prepared prior to assay procedure. All reagents were provided with Quantikine® immunoassay kit.

3.3.1.III Rat IL-6, IL-1β, TNF-α and IFN-γ control

The control provided with kit, was reconstituted with 1 ml double distilled water.

3.3.1.IV Rat IL-6 IL-1β, TNF-α and IFN-γ conjugate concentrate

For 96 wells

Conjugate concentrate 0.5 ml

Conjugate diluent 11 ml

3.3.1.V Washing buffer

For 96 wells

Washing buffer concentrate 25 ml

dd H2O to 625 ml

3.3.1.VI Substrate solution

Equal volumes of color reagents A and B, provided by kit, were mixed together, and solution was used with 15 min.

3.3.1.VII Standard and sample preparation

[page 51]

The standards were reconstituted with 2 ml of calibrator diluent. This stock solution (2000 pg/ml) was used to prepare a serial dilution ranging from 31.2 pg/ml to 2000 pg/ml. Calibrator diluent served as zero standard. Prior to assay, serum samples were diluted 2 fold into calibration diluent.

Anmerkungen

The source is not given.

Sichter
(Hindemith) Schumann

[12.] Iam/Fragment 047 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:01:29 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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3.3.3.g. Assay procedure

The whole procedure was performed at room temperature. All samples, standards and controls were brought to the RT and assayed in duplicates. To synchronize the reaction in each well, all reagents were pipette using a multi-channel pipette. 50 μl of assay diluent was added to each well. Standards, control and samples were added in a quantity of 50 μl per well. The components were mixed by gentle tapping the plate frame for 1 min. After that, the plate was covered with the adhesive strip provided and incubated for 2 h at room temperature. After the incubation period each well was aspirated and washed with 400 μl of wash buffer using a manifold dispenser, and procedure was repeated four times for a total of five washes. After washing, 100 μl of rat CXCL2 conjugate was added to each well. The plate was covered with a new adhesive strip and incubated for another 2 h at room temperature. The aspiration and washing procedure was performed as described above. Subsequently, 100 μl of substrate solution was added to each well to start enzymatic reaction and plate was incubated in the dark for 30 min at room temperature. To stop the enzymatic reaction, 100 μl of stop solution was added to each well, followed by determination of optical density of each well using a microplate reader (Dynatech Laboratories) set to dual wavelength mode (test filter –450 nm, reference filter –570 nm). The calculation of results was performed with a program (Dynatech MRX software, version 1.33) created in accordance to the manual instructions (Quantikine® immunoassay kit).

3.4 Methods in clinical chemistry

3.4.1 Transaminases

Transminases (ALT and AST) are the most important representatives of a group of enzymes, the aminotransferases or transaminases, which catalyze the conversion of α-keto acids into amino acids by transfer of amino groups. As a liver specific enzyme AST is significantly elevated in hepatobiliary disease, increased ALT levels, however can occur in connection with damages of heart or skeletal muscle as well as of liver parenchyma. Serum level of transaminases (AP, ALT and AST) were determined by routine clinical laboratory test using diasys kit (diagnostic systems international Holzheim Germany)

[Page 51]

3.3.1.VIII Assay procedure

The whole procedure was performed at room temperature. All samples, standards and controls were brought to the RT and assayed in duplicates. To synchronize the reaction in each well, all reagents were pipette using a multi-channel pipette. 50 μl of assay diluent was added to each well. Standards, control and samples were added in a quantity of 50 μl per well. The components were mixed by gentle tapping the plate frame for 1 min. After that, the plate was covered with the adhesive strip provided and incubated for 2 h at room temperature. After the incubation period each well was aspirated and washed with 400 μl of wash buffer using a manifold dispenser, and procedure was repeated four times for a total of five washes. After washing, 100 μl of rat IL-6, IL-1β, TNF-α or IFN γ conjugate was added to each well. The plate was covered with a new adhesive strip and incubated for another 2 hours at room temperature. The aspiration and washing procedure was performed as described above. Subsequently, 100 μl of substrate solution was added to each well to start enzymatic reaction and plate was incubated in the dark for 30 min at room temperature. To stop the enzymatic reaction, 100 μl of stop solution was added to each well, followed by determination of optical density of each well using a microplate reader (Dynatech Laboratories) set to dual wavelength mode (test filter 450 nm, reference filter 570 nm). The calculation of results was performed with a program (Dynatech MRX software, version 1.33) created in accordance to the manual instructions (Quantikine® immunoassay kit).

[Page 54]

3.4 Methods in clinical chemistry

[Page 56]

3.4.2 Transaminases

Transminases (ALT and AST) are the most important representatives of a group of enzymes, the aminotransferases or transaminases, which catalyze the conversion of α- keto acids into amino acids by transfer of amino groups. As a liver specific enzymes ALT is significantly elevated in hepatobiliary disease, increased AST levels however, can occur in connection with damages of heart or skeletal muscle as well as of liver parenchyma. [...] Serum level of transaminases were determined by routine clinical laboratory test using diasys kit (diagnostic systems international Holzheim Germany)

Anmerkungen

But for minutiae (that adapt the existing text to Iam's research subject) identical, nevertheless not marked as a citation.

Sichter
(Graf Isolan) Schumann

[13.] Iam/Fragment 048 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:51 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

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3.4.2 Statistical Analysis

The data were analyzed using Prism Graph pad 4 software (San Diego, USA). All experimental errors are shown as S.E.M. Statistical significance was calculated by Student’s t test and one way ANOVA and Dunnett post hoc test. Significance was accepted at P < 0.05.

3.4.3 Safety Measures

All operations with genetically modified organisms and plasmid DNA were performed in accordance to the ‘‘Gentechnikgesetz’’ of 1990 and to the rules prescribed by the ‘‘Gentechnik-Sicherheitsverordnung’’ of 1990. Ethidium bromide, formaldehyde, DEPC and other chemicals deleterious for the environment, when used in the course of the work, were carefully managed and disposed properly in accordance with institutional guidelines. All the operations with radioactive chemicals were performed in a radioactivity class II laboratory and the radioactive waste was disposed off according to the institutional instructions.

3.5 Statistical analysis

The data were analysed using Prism Graph pad 4 software (San Diego, USA). All experimental errors are shown as SEM. Statistical significance was calculated by Student’s t test and one way ANOVA with Dunnett post hoc test. Significance was accepted at P < 0.05.

3.6 Safety measures

All operations with genetically modified organisms and plasmid DNA were performed in accordance to the ‘‘Gentechnikgesetz’’ of 1990 and to the rules prescribed by the ‘‘Gentechnik-Sicherheitsverordnung’’ of 1990. Ethidium bromide, formaldehyde, DEPC and other chemicals deleterious for the environment, when used in the course of the work, were carefully managed and disposed properly in accordance with institutional guidelines. All the operations with radioactive chemicals were performed in a radioactivity class II laboratory and the radioactive waste was disposed off according to the institutional instructions.

Anmerkungen

Identical, nevertheless not marked as a citation.

Sichter
(Graf Isolan) Schumann

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