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6 gesichtete Fragmente: "Verdächtig" oder "Keine Wertung"

[1.] Sj/Fragment 030 02 - Diskussion
Bearbeitet: 3. December 2016, 17:38 (WiseWoman)
Erstellt: 26. November 2016, 14:25 LieschenMueller
Fragment, Gesichtet, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel sysop, Sj

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KeineWertung
Bearbeiter
LieschenMueller
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 30, Zeilen: 1 - 11
Quelle: Palmada et al 2006
Seite(n): 422, Zeilen: r.col:13ff
Isolation of mouse adipocytes

Adipocytes were isolated from epididymal fat pads of 129/SvJ mice by collagenase digestion as described previously (117). Epididymal fat pads were minced and digested with 2 mg/ml type II collagenase for 1h at 37°C in Krebs Ringer bicarbonate Hepes buffer (KRBH: 120 mmol/l NaCl, 4 mmol/l KH2PO4, 1 mmol/l MgSO4, 1 mmol/l CaCl2, 10 mmol/l NaHCO3, 200 nmol/l adenosine, 30 mmol/l Hepes, pH 7.4) containing 1 % fraction V BSA. The resulting cell suspension was filtered through a nylon mesh (250 μm) and washed 3 times with KRBH buffer containing 3 % BSA. Then adipocytes were resuspended in KRBH buffer with 3 % BSA. An aliquot of the final cellular suspension was taken to measure lipocrit and cell number.


117. Palmada,M, Boehmer,C, Akel,A, Rajamanickam,J, Jeyaraj,S, Keller,K, Lang,F: SGK1 Kinase Upregulates GLUT1 Activity and Plasma Membrane Expression. Diabetes 55:421-427, 2006

Isolation of mice adipocytes. Adipocytes were isolated from 129/SvJ mice epididymal fat pads by collagenase digestion as previously described (35). The fat pads were minced and digested with 2 mg/ml type II collagenase for 1 h at 37°C in Krebs Ringer bicarbonate HEPES buffer (120 mmol/l NaCl, 4 mmol/l KH2PO4 , 1 mmol/l MgSO4 , 1 mmol/l CaCl2 , 10 mmol/l NaHCO3 , 200 nmol/l adenosine, and 30 mmol/l HEPES, pH 7.4) containing 1% fraction V BSA. The resulting cell suspension was filtered through a nylon mesh (250 m) and washed three times with Krebs Ringer bicarbonate HEPES buffer containg 3% BSA. Then adipocytes were resuspended in Krebs Ringer bicarbonate HEPES buffer with 3% BSA. An aliquot of the final cellular suspension was taken to measure lipocrit and cell number.

35. Ruan H, Zarnowski MJ, Cushman SW, Lodish HF: Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes. J Biol Chem 278: 47585–47593, 2003

Anmerkungen

A source for the method is named, but it is not made clear that much more is taken from this source, of which Sj is co-author.

Sichter
(LieschenMueller), WiseWoman

[2.] Sj/Fragment 031 10 - Diskussion
Bearbeitet: 3. December 2016, 21:32 (WiseWoman)
Erstellt: 26. November 2016, 22:53 LieschenMueller
Fragment, Gesichtet, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel, Sj

Typus
KeineWertung
Bearbeiter
LieschenMueller
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 31, Zeilen: 10
Quelle: Palmada et al 2006
Seite(n): 422, Zeilen: r.col:18f
Tritium-labeled 2-deoxy-D-glucose (2-DOG) was used as the glucose analogue for uptake determination. Groups of 10-15 oocytes injected with GLUT4 were incubated for 2 h with 1 μM insulin. Thereafter oocytes were placed in 0.25 ml of ND96 (96 mmol/l NaCl, 2 mmol/l KCl, 1.8 mmol/l CaCl2, 1 mmol/l MgCl2 and 5 mmol/l HEPES, pH 7.4) containing 37 KBq 3H 2-DOG and 50 μmol/l (or the indicated amount for kinetic analysis) of unlabeled 2-DOG. After incubation for 10 min at room temperature with 2-DOG (linear range of uptake), uptake was terminated by washing the oocytes four times with 3 ml of ice-cold phosphate- buffered saline (PBS) containing 100 mmol/l 2-DOG. Oocytes were individually transferred into scintillation vials and dissolved by adding 200 μl of 10% SDS before radioactivity was determined.

In HEK-293 cells, 2-DOG uptake was measured 2 days after transfection by incubating cells at 37°C for 5 min (linear range of uptake) in glucose free krebs Ringer HEPES buffer containing 3H 2-DOG 0.1 μCi/well and 0.3 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretein. Uptake was terminated by rapid aspiration of uptake solution and washing four times with ice-cold PBS containing 50 mmol/l unlabeled 2-DOG. Thereafter, cells were lyses [sic] with 10mmol/l NaOH/0.1%/Triton-X-100, and radioactivity incorporated into the cells was measured with a liquid scintillation counter. Protein concentrations were determined by the Bradford method. For kinetic analyis [sic], 2-DOG uptake was measured by incubating cells with 3H 2-DOG 0.1 μCi/well and various concentrations of unlabeled 2-DOG at 37°C for 5 minutes.

In isolated adipocytes, 2-DOG uptake was measured by incubating the cells with 0.1 μCi 3H 2-DOG and 0.1 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretin.

Transport studies. Tritium-labeled 2-deoxy-D-glucose (2-DOG) was used as the glucose analog for uptake determination. In Xenopus oocytes, the transport assay was performed 4 days after cRNA injection and contained 10–15 single oocytes in 0.25 ml ND96 (96 mmol/l NaCl, 2 mmol/l KCl, 1.8 mmol/l CaCl2, 1 mmol/l MgCl2, and 5 mmol/l HEPES, pH 7.4) containing 1 μCi 3H 2-DOG and 50 μmol/l (or the indicated amount in the kinetic analysis) of unlabeled 2-DOG. After incubation for 30 min at room temperature with

[page 423]

2-DOG (linear range of uptake), uptake was terminated by washing the oocytes four times with 3 ml ice-cold PBS containing 100 mmol/l unlabeled 2-DOG. Oocytes were individually transferred into scintillation vials and dissolved by adding 200 μl of 10% SDS before the radioactivity was determined. In HEK-293 cells, 2-DOG uptake was measured 2 days after transfection by incubating the cells at 37°C for 5 min (linear range of uptake) in glucose-free Krebs Ringer HEPES buffer containing 3H 2-DOG 0.1 μCi/well and 0.3 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretin. Uptake was terminated by rapid aspiration of uptake solution and washing four times with ice-cold PBS containing 50 mmol/l unlabeled 2-DOG. Thereafter, cells were lysed with 10 mmol/l NaOH/0.1% Triton X-100, and radioactivity incorporated into the cells was measured with a liquid scintillation counter. Protein concentrations were determined by the Bradford method. For kinetic analysis, 2-DOG uptake was measured by incubating the cells with 3H 2-DOG 0.1 Ci/well and various concentrations of unlabeled 2-DOG at 37°C for 5 min. In isolated adipocytes, 2-DOG uptake was measured by incubating the cells with 0.1 μCi 3H 2-DOG and 0.1 mmol/l cold 2-DOG with or without 0.1 mmol/l phloretin.

Anmerkungen

The source was last referenced on Sj/Fragment_030_02. Sj is co-author of the source. The extent of the textual correspondence is not made clear.

Sichter
(LieschenMueller), WiseWoman

[3.] Sj/Fragment 032 01 - Diskussion
Bearbeitet: 3. December 2016, 17:47 (WiseWoman)
Erstellt: 26. November 2016, 23:27 LieschenMueller
Fragment, Gesichtet, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel sysop, Sj

Typus
KeineWertung
Bearbeiter
LieschenMueller
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 32, Zeilen: 4-19
Quelle: Palmada et al 2006
Seite(n): 423, Zeilen: l. col:18ff; r.col:9ff
[Uptake was] terminated after 5 min by centrifugation of cells through dinonylphthalate. The separated cells were removed from the top of the oil layer, and cell-associated radioactivity was quantified.

3.7 Western blotting

To determine whole-cell GLUT1 and SGK1 expression in HEK-293 and adipocytes, cells were homogenized in lysis buffer and 30 μg protein were separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% non fat dry milk in PBS/0.15% Tween-20 for 1 h at room temperature, blots were incubated overnight at 4°C with a goat anti- GLUT1 antibody (diluted 1:100 in PBS/0.15% Tween 20/5% nonfat dry milk; Santa Cruz Biotechnology), a rabbit anti-SGK1 antibody (diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk; Upstate, Dundee, U.K.), or a rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) horseradish peroxidase conjugated antibody (diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk). GAPDH was used to demonstrate equal protein loading. Secondary peroxidase-conjugated donkey anti-goat IgG antibody (diluted 1:2000 in PBS/0.15% Tween 20/5% nonfat dry milk; Santa Cruz Biotechnology) or sheep anti-rabbit IgG antibody (diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk; Amersham, Freiburg, Germany) was used for chemiluminescent detection of GLUT1 or SGK1 with an enhanced chemiluminescence kit (Amersham), respectively. Band intensities were quantified using Quantity One Analysis software (Biorad, Munich, Germany).

Uptake was terminated after 5 min by centrifugation of cells through dinonylphthalate. The separated cells were removed from the top of the oil layer, and cell-associated radioactivity was quantified. [...]


Western blotting. To determine whole-cell GLUT1 and SGK1 expression in oocytes, transfected HEK-293 cells, and adipocytes, cells were homogenized in lysis buffer, and 30 μg protein were separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk in PBS/0.15% Tween 20 for 1 h at room temperature, blots were incubated overnight at 4°C with a goat anti-GLUT1 antibody (diluted 1:100 in PBS/0.15% Tween 20/5% nonfat dry milk; Santa Cruz Biotechnology), a rabbit anti-SGK1 antibody (diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk; Upstate, Dundee, U.K.), or a rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) horseradish peroxidase–conjugated antibody (diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk). GAPDH was used to demonstrate equal protein loading. Secondary peroxidase-conjugated donkey anti-goat IgG antibody (diluted 1:2000 in PBS/0.15% Tween 20/5% nonfat dry milk; Santa Cruz Biotechnology) or sheep anti-rabbit IgG antibody (diluted 1:1,000 in PBS/0.15% Tween 20/5% nonfat dry milk; Amersham, Freiburg, Germany) was used for chemiluminescent detection of GLUT1 or SGK1 with an enhanced chemiluminescence kit (Amersham), respectively. Band intensities were quantified using Quantity One Analysis software (Biorad, Munich, Germany)

Anmerkungen

The source was last referenced on Sj/Fragment_030_02. Sj is co-author of the source. The extent of the textual correspondence is not made clear.

Sichter
(LieschenMueller), WiseWoman

[4.] Sj/Fragment 041 01 - Diskussion
Bearbeitet: 3. December 2016, 17:50 (WiseWoman)
Erstellt: 27. November 2016, 13:46 LieschenMueller
Fragment, Gesichtet, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel, Sj

Typus
KeineWertung
Bearbeiter
LieschenMueller
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 41, Zeilen: 1ff
Quelle: Palmada et al 2006
Seite(n): 424, Zeilen: 1: Bildunterschrift FIG.6
4.6 S422DSGK1 enhances 2-DOG transport in HEK-293 cells without affecting total protein expression levels of GLUT1

It was already observed the upregulation of GLUT1 by SGK1 in Xenopus oocytes. GLUT1 modulation by SGK1 was observed in mammalian cells (HEK-293). HEK-293 cells were transfected with pIRES2EGFP-S422DSGK1, pIRES2EGFPK127NSGK1 or with empty vector (as a control) and two days later 2-DOG uptake was measured in the presence and absence of GLUT1 inhibitor phloretin (0.1 mM).

FIG. 6. S422DSGK1 enhances 2-DOG transport in HEK-293 cells without affecting total GLUT1 expression levels. HEK-293 cells were transfected with pIRES2EGFP-S422DSGK1, pIRES2EGFP-K127NSGK1, or empty vector, and 2 days later labeled 2-DOG uptake was studied in the presence and absence of GLUT1 inhibitor 0.1 mmol/l phloretin (A)....
Anmerkungen

The source was last referenced on Sj/Fragment_030_02. Sj is co-author of the source.

Sichter
(LieschenMueller), WiseWoman

[5.] Sj/Fragment 041 00 - Diskussion
Bearbeitet: 3. December 2016, 21:42 (WiseWoman)
Erstellt: 28. November 2016, 09:52 LieschenMueller
Fragment, Gesichtet, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel, Sj

Typus
KeineWertung
Bearbeiter
LieschenMueller
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 41, Zeilen: Fig.15 und Bildunterschrift
Quelle: Palmada et al 2006
Seite(n): 424, Zeilen: Fig.6A; Bildunterschrift Z.1-5
Dsa 043 diss.png

Figure 15. S422DSGK1 enhances 2-DOG transport in HEK-293 cells without affecting total GLUT1 expression levels. HEK-293 cells were transfected with pIRES2EGFP-S422DSGK1, pIRES2EGFPK127NSGK1 or empty vector and 2 days later, labeled 2-DOG uptake was studied in the presence and absence of GLUT1 inhibitor 0.1 mM phloretin

Dsa 043 source.png

FIG. 6. S422DSGK1 enhances 2-DOG transport in HEK-293 cells without affecting total GLUT1 expression levels. HEK-293 cells were transfected with pIRES2EGFP-S422DSGK1, pIRES2EGFP-K127NSGK1, or empty vector, and 2 days later labeled 2-DOG uptake was studied in the presence and absence of GLUT1 inhibitor 0.1 mmol/l phloretin (A)...

Anmerkungen

The source was last referenced on Sj/Fragment_030_02. Sj is co-author of the source. The extent of the textual correspondence is not made clear.

Sichter
(LieschenMueller), WiseWoman

[6.] Sj/Fragment 042 01 - Diskussion
Bearbeitet: 3. December 2016, 21:22 (WiseWoman)
Erstellt: 28. November 2016, 14:32 LieschenMueller
Fragment, Gesichtet, KeineWertung, Palmada et al 2006, SMWFragment, Schutzlevel, Sj

Typus
KeineWertung
Bearbeiter
LieschenMueller
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 42, Zeilen: 1ff (entire page)
Quelle: Palmada et al 2006
Seite(n): 424; 425, Zeilen: 424 r.col: 44f ; l.col: Bildunterschrift Zeile 6; 425 l.col: 1f
[Tracer-flux studies revealed an increase in GLUT1 transport rate (from 2.3 ± 0.6nmol/min/mg protein, n = 3, to 5.1 ± 0.6 nmol/min/mg protein, n = 3, Figure 15) upon] coexpression of S422DSGK1. As observed in Xenopus oocytes, GLUT1 upregulation by SGK1 was dependent on the catalytic activity of the kinase as the effect was lacking upon expression of the catalytical inactive mutant K127NSGK1 (from 2.3 ± 0.6nmol/min/mg protein, n = 3, to 1.8 ± 0.2 nmol/min/mg protein, n = 3).

Sj-Diss-042 01.png

Figure 16. Western blots of whole cell lysates were performed . Arithmetic means ± SEM. * indicates statistically significant difference to uptake in HEK-293 cells transfected with the empty vector (*p < 0.05).

Western blotting of whole cell lysates indicated that the kinase fails to induce GLUT1 protein synthesis (Figure 16). For western blotting, GLUT1/GAPDH band intensities from three independent experiments were normalized in each transfection to the value of GLUT1/GAPDH band intensity of oocytes expressing GLUT1 alone.

Sj-Quelle-042 01.png

Fig. 6. [...] Western blots of whole-cell lysates were performed (B). Arithmetic means ± SE. *Statistically significant difference to uptake in HEK-293 cells transfected with the empty vector (*P < 0.05). For Western blotting, GLUT1/GAPDH band intensities from three independent experiments were normalized in each transfection to the value of GLUT1/ GAPDH band intensity of oocytes expressing GLUT1 alone.

[...]

Tracer-flux studies revealed an increase in GLUT1 transport rate (from 2.3 ± 0.6 [n = 3] to 5.1 ± 0.6 nmol • min-1 • mg protein-1 [n = 3], Fig. 6A) upon coexpression of S422DSGK1. As observed in Xenopus oocytes, GLUT1 upregulation by SGK1 was dependent on the catalytic activity of the kinase, as the effect was lacking upon expression of the catalytical inactive mutant K127NSGK1 (from 2.3 ± 0.6 [n = 3] to 1.8 ± 0.2 nmol • min-1 • mg protein-1 [n = 3]).

[page 425]

Western blotting of whole-cell lysates indicated that the kinase fails to induce GLUT1 protein synthesis (Fig. 6B).

Anmerkungen

The source was last referenced on Sj/Fragment_030_02. Sj is co-author of the source.

Sichter
(LieschenMueller), WiseWoman

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