Divide and conquer: Rat liver tissue proteomics based on the analysis of purified constituents

Teufelhofer, Olga; Parzefall, Wolfram; Elbling, Leonilla; Kainzbauer, Eveline; Grasl‐Kraupp, Bettina; Zielinski, Christoph; Schulte‐Hermann, Rolf; Gerner, Christopher

doi:10.1002/elps.200600017

Cited by 8 publications

(9 citation statements)

References 29 publications

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“…Therefore, an improved understanding of non-genotoxic compounds has to consider drug effects on stroma cells, even if these cells are not transformed to cancer cells. In order to assign molecular events caused by NGCs to the different cell types of the liver, we isolated primary cells by liver perfusion and separated them into parenchymal HCs and NPCs as described previously [54,55]. Here, we isolated cells from untreated and PB-treated animals in order to investigate in vivo effects.…”

Section: Discussionmentioning

confidence: 99%

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

et al. 2013

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Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

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Section: Discussionmentioning

confidence: 99%

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

et al. 2013

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“…1‐D gels were stained over night with colloidal blue stain (Invitrogen). 2‐DE was performed essentially as described earlier 11, 12 on a Protean II xi electrophoresis system (Bio‐Rad, Hercules, CA, USA). Samples equivalent to 300 μg protein were mixed with four volumes of methanol, one volume of chloroform, and three volumes of water.…”

Section: Methodsmentioning

confidence: 99%

“…Following 12 h rehydration of IPG strips (17 cm, pH 5–8; Bio‐Rad) at room temperature, IEF was carried out in a stepwise mode (1 h 0–500 V linear; 5 h 500 V; 5 h 500–3500 V linear; 12 h 3500 V). SDS‐PAGE and staining with a 400 nM solution of ruthenium II tris bathophenanthroline disulfonate was performed as published 12, 13. Fluorography was performed on a FluorImager 595 (GE Healthcare) at a resolution of 100 μm.…”

Section: Methodsmentioning

confidence: 99%

Quantitative assessment of human serum high‐abundance protein depletion

et al. 2008

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The aim of this study is to quantify the effectivity of the depletion of human high-abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2-DE and bottom-up shotgun MS combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high-abundance protein depletion. Nano-liquid chromatography linked MS allowed the efficient identification of several low-abundant proteins that were not identified on the 2-DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.

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“…Most proteomic research of liver fibrosis has been performed using tissues, plasma, or cell lines. There are few studies of the proteome in primary cells, especially NPCs such as KCs [17,18], LSECs [19], and hepatic stellate cells [20]. For example, Hirsch et al [18] investigated the effect of ischemia/ reperfusion injury on the proteome of KCs from a rat model of ischemia/reperfusion injury, and found elevation of Cu, Zn-superoxide dismutase.…”

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confidence: 99%

“…For example, Hirsch et al [18] investigated the effect of ischemia/ reperfusion injury on the proteome of KCs from a rat model of ischemia/reperfusion injury, and found elevation of Cu, Zn-superoxide dismutase. Teufelhofer et al [17] analyzed the proteomes of purified rat liver primary cells and blood plasma for comparison with that of liver whole tissue. Li et al [19] reported the proteomic profile of the plasma membrane of LSECs from normal rat liver.…”

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confidence: 99%

Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats

Zhao

Feng

Jia

et al. 2014

Sci. China Life Sci.

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Elucidation of the mechanisms of liver fibrogenesis is important to treat liver fibrosis. In this study, we established rat models of liver fibrosis with stages from 0-1, 2, and 3-4 to 4 at 2, 4, 6, and 8 weeks, respectively, by injection of pig serum. Liver fibrogenesis was detected by Masson's trichrome staining. Rat non-parenchymal cells (NPCs) were enriched 4-fold by Percoll density gradient centrifugation. Protein extracts from NPCs were prepared at 4 and 8 weeks, separated by two-dimensional electrophoresis, and then stained with Coomassie Blue G-250. At 4 weeks, we identified 18 non-redundant differentially expressed proteins of which protein disulfide-isomerase associated protein 3 (PDIA3) and NDUV showed consistent expression at protein and mRNA levels from 4 to 8 weeks. PDIA3 was found to be down-regulated by Western blotting in the rat model and immunohistochemically in human liver. Our results revealed important aspects of the pathogenesis/progression of liver fibrosis and demonstrated important changes in protein expression levels of NPCs at various stages of liver fibrosis. liver fibrosis, proteomics, non-parenchymal cells, protein disulfide-isomerase associated protein 3 Citation:Zhao QQ, Feng YL, Jia XF, Yin L, Zheng Y, Ouyang DS, Zhou HH, Zhang LJ. Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats.

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Divide and conquer: Rat liver tissue proteomics based on the analysis of purified constituents

Cited by 8 publications

References 29 publications

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

Quantitative assessment of human serum high‐abundance protein depletion

Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats

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