[show abstract][hide abstract] ABSTRACT: Nonalcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which carries a significant risk of progression to cirrhosis and hepatocellular carcinoma. Since NASH is a progressive but reversible condition, it is desirable to distinguish NASH from simple steatosis, and to treat NASH patients at an early stage. To establish appropriate diagnosis and therapy, the pathological mechanisms of the disease should be elucidated; however, these have not been fully clarified for both NASH and simple steatosis. This study aims to reveal the differences between simple steatosis and NASH.
This study used fatty liver Shionogi (FLS) mice as a NASH model, for comparison with dd Shionogi (DS) mice as a model of simple steatosis. Genome-wide gene expression analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Array, which contains 45101 probe sets for known and predicted genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to investigate gene expression changes and protein localizations.
DNA microarray analysis of the liver transcriptomes and qRT-PCR of both types of mice revealed that LCN2, CXCL1 and CXCL9 mRNAs were overexpressed in FLS mouse livers. Immunohistochemistry showed that CXCL1 protein was mainly localized to steatotic hepatocytes. CXCL9 protein-expressing hepatocytes and sinusoidal endothelium were localized in some areas of inflammatory cell infiltration. Most interestingly, hepatocytes expressing LCN2, a kind of adipokine, were localized around almost all inflammatory cell clusters. Furthermore, there was a positive correlation between the number of LCN2-positive hepatocytes in the specimen and the number of inflammatory foci.
Overexpression and distinct localization of LCN2, CXCL1 and CXCL9 in the liver of fatty liver Shionogi mice suggest significant roles of these proteins in the pathogenesis of NASH.
[show abstract][hide abstract] ABSTRACT: Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, MMP-9 and LCN2, which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP-9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC-MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: The mechanism of alcohol-induced pancreatic damage is unclear. The aim of this study was to clarify the effects of chronic alcohol intake on the pancreatic proteome.
Rats were fed an alcohol-containing Lieber-DeCarli liquid diet, and the pancreatic proteome was compared with that of pair-fed control rats using agarose 2-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry.
The expression of 3 proteins was consistently altered in alcohol-fed rats: 1 protein was down-regulated, and 2 proteins were up-regulated. The 2 up-regulated proteins were identified as 2,4-dienoyl-CoA reductase and hydroxymethylglutaryl-CoA synthase (HMGCS2). The combined concentration of malondialdehyde and 4-hydroxyalkenals was significantly greater in alcohol-fed rats. It is noteworthy that the reactivity of anti-4-hydroxy-2-nonenal antibody was significantly higher toward HMGCS2 isolated from alcohol-fed rats. The activity of HMGCS2 was higher in alcohol-fed rats, but the relative increase in enzyme activity in alcohol-fed rats was less than the relative increase in HMGCS2 expression.
Chronic alcohol consumption results in distinct alterations in the expression of 3 pancreatic proteins. The reactivity of 4-hydroxy-2-nonenal toward one of the up-regulated proteins, HMGCS2, increased markedly following chronic alcohol intake, suggesting that up-regulation of HMGCS2 is connected with alterations of lipid peroxidation induced by alcohol.
[show abstract][hide abstract] ABSTRACT: LC-ESI/MS/MS-based shotgun proteomics is currently the most commonly used approach for the identification and quantification of proteins in large scale studies of biomarker discovery. In the past several years, the shotgun proteomics technologies have been refined toward further enhancement of proteome coverage. In the complex series of protocols involved in shotgun proteomics, however, loss of proteolytic peptides during the lyophilization step prior to the LC/MS/MS injection has been relatively neglected despite the fact that the dissolution of the hydrophobic peptides in lyophilized samples is difficult in 0.05-0.1% TFA or formic acid, causing substantial loss of precious peptide samples. In order to prevent the loss of peptide samples during this step we devised a new protocol by use of Invitrosol (IVS), a commercially available surfactant compatible with ESI-MS; by dissolving the lyophilized peptides by IVS, we show improved recovery of hydrophobic peptides, leading to enhanced coverage of proteome. Thus, the use of IVS in the recovery step of lyophilized peptides will help the shotgun proteomics analysis by expanding the proteome coverage, which would significantly promote the discovery and development of new diagnostic markers and therapeutic targets.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Proteomic approaches may provide new insights into pathological conditions associated with alcoholism. The aim of this study was to conduct a proteomic analysis of liver tissue and serum in chronically alcohol-fed rats using agarose 2-dimensional gel electrophoresis (2-DE) and 3-step serum proteome analysis. METHODS: A total of 12 rats were pair-fed nutritionally adequate liquid diet containing ethanol as 36% of the total energy or an isocaloric control diet for 2 months. Rat liver homogenates and cytosol fractions were subjected to agarose 2-DE. Serum samples were subjected to 3-step serum proteome analysis involving immunodepletion of abundant proteins followed by fractionation using reverse-phase high-performance liquid chromatography and 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Candidate proteins were digested with trypsin and identified using mass spectrometry. Observed differences in protein expression levels were confirmed using Western blotting. RESULTS: A total of 46 protein spots were found to be differentially expressed in the liver homogenates and cytosol fractions of alcohol-fed rats relative to pair-fed controls. The most notable change was down-regulation of a 29-kDa protein, which was subsequently identified as carbonic anhydrase III (CA III). Down-regulation of this protein in alcohol-fed rats was confirmed by Western blotting. The messenger RNA level of CA III was decreased as well. In rat serum, a total of 41 proteins were differentially expressed. Of these proteins, only betaine-homocysteine methyltransferase (BHMT) was also found to be differentially expressed in the liver. CONCLUSIONS: A combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats revealed that the expression of CA III is significantly down-regulated in the liver of alcohol-fed rats. Our results also showed that BHMT expression is up-regulated in both the liver and serum of alcohol-fed rats.
Alcoholism Clinical and Experimental Research 10/2012; · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: In Helicobacter pylori infection, vacuolating cytotoxin (VacA)-induced mitochondrial damage leading to apoptosis is believed to be a major cause of cell death. It has also been proposed that VacA-induced autophagy serves as a host mechanism to limit toxin-induced cellular damage. Apoptosis and autophagy are two dynamic and opposing processes that must be balanced to regulate cell death and survival. Here we identify the low-density lipoprotein receptor-related protein-1 (LRP1) as the VacA receptor for toxin-induced autophagy in the gastric epithelial cell line AZ-521, and show that VacA internalization through binding to LRP1 regulates the autophagic process including generation of LC3-II from LC3-I, which is involved in formation of autophagosomes and autolysosomes. Knockdown of LRP1 and Atg5 inhibited generation of LC3-II as well as cleavage of PARP, a marker of apoptosis, in response to VacA, whereas caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone (Z-VAD-fmk), and necroptosis inhibitor, Necrostatin-1, did not inhibit VacA-induced autophagy, suggesting that VacA-induced autophagy via LRP1 binding precedes apoptosis. Other VacA receptors such as RPTPα, RPTPβ, and fibronectin did not affect VacA-induced autophagy or apoptosis. Therefore, we propose that the cell surface receptor, LRP1, mediates VacA-induced autophagy and apoptosis.
Journal of Biological Chemistry 07/2012; 287(37):31104-15. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Although gemcitabine has been widely used as a first-line chemo reagent for patients with pancreatic cancer, the response rate remains low. We previously identified Annexin II as a factor involved in gemcitabine resistance against pancreatic cancer. The aims of this study were to elucidate the signaling mechanism by which Annexin II induces gemcitabine resistance and to develop a new therapy that overcomes the resistance against gemcitabine. METHODS: We compared the specific profiles of 12 targeted phosphorylated (p-) signaling proteins in gemcitabine-resistant (GEM-) and its wild-type pancreatic cancer cell lines (MIA PaCa-2) using the Bio-Plex assay system. We also evaluated the expression levels of Annexin II and two phosphoproteins, which showed different expressions in these two cell lines, by immunohistochemistry. RESULTS: Annexin II overexpression was significantly associated with rapid recurrence after gemcitabine-adjuvant chemotherapy in patients with resected pancreatic cancer (P < 0.05). Bio-Plex analysis showed up-regulation of p-Akt in GEM-MIA PaCa-2 cells in which Annexin II is highly expressed. The expression level of p-Akt was significantly correlated with that of the downstream protein, p-mTOR, in pancreatic cancer tissues. Inhibition of mTOR phosphorylation canceled gemcitabine resistance in GEM-MIA PaCa-2 cells. CONCLUSIONS: The Akt/mTOR pathway is involved in mechanisms of gemcitabine resistance induced by Annexin II in pancreatic cancer cells. This indicates that combination therapy with the mTOR inhibitor may overcome gemcitabine resistance. Annexin II as an indicator for selection of gemcitabine resistance could thus be applied to the development of novel tailor-made approaches for pancreatic cancer treatment.
Journal of Surgical Research 06/2012; · 2.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.
[show abstract][hide abstract] ABSTRACT: The Far UpStream Element (FUSE)-binding protein-interacting repressor (FIR), a c-myc transcriptional suppressor, is alternatively spliced removing the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. SAP155 is a subunit of the essential splicing factor 3b (SF3b) subcomplex in the spliceosome. This study aims to study the significance of the FIR-SAP155 interaction for the coordination of c-myc transcription, pre-mRNA splicing, and c-Myc protein modification, as well as to interrogate FIRΔexon2 for other functions relating to altered FIR pre-mRNA splicing. Knockdown of SAP155 or FIR was used to investigate their reciprocal influence on each other and on c-myc transcription, pre-mRNA splicing, and protein expression. Pull down from HeLa cell nuclear extracts revealed the association of FIR, FIRΔexon2, and SF3b subunits. FIR and FIRΔexon2 were coimmunoprecipitated with SAP155. FIR and FIRΔexon2 adenovirus vector (Ad-FIR and Ad-FIRΔexon2, respectively) were prepared to test for their influence on c-myc expression. FIR, SAP155, SAP130, and c-myc were coordinately upregulated in human colorectal cancer. These results reveal that SAP155 and FIR/FIRΔexon2 form a complex and are mutually upregulating. Ad-FIRΔexon2 antagonized Ad-FIR transcriptional repression of c-myc in HeLa cells. Because FIRΔexon2 still carries RRM1 and RRM2 and binding activity to FUSE, it is able to displace repression competent FIR from FUSE in electrophoretic mobility shift assays, thus thwarting FIR-mediated transcriptional repression by FUSE. Thus aberrant FIRΔexon2 production in turn sustained c-Myc expression. In conclusion, altered FIR and c-myc pre-mRNA splicing, in addition to c-Myc expression by augmented FIR/FIRΔexon2-SAP155 complex, potentially contribute to colorectal cancer development.
Molecular Cancer Research 04/2012; 10(6):787-99. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.
Journal of proteomics 03/2012; 75(6):1803-15. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: rohibitin is localized to mitochondria where it might have a role in the maintenance of mitochondrial function and protection against senescence. In this study, we show that prohibitin is up-regulated in lung squamous cell carcinoma tissues compared with adjacent normal tissues using agarose 2-dimensional differential gel electrophoresis and immunoblotting. Prohibitin expression was further evaluated by immunohistochemistry. We statistically analyzed the association of prohibitin expression with clinicopathologic indicators in 78 patients with lung squamous cell carcinoma. Our data suggested that prohibitin expression was positively correlated with the International Union Against Cancer (UICC) classification of tumor grade (P < .001), pathologic stage (P < .001), tumor size (P = .01), and lymph node metastasis (P = .004). Furthermore, we found that prohibitin expression was an independent prognostic indicator (P = .037) for overall survival of patients with lung squamous cell carcinoma by multivariate analysis using the Cox regression method. These findings may encourage further studies investigating prohibitin function in lung squamous cell carcinoma.
Human pathology 02/2012; 43(8):1282-8. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Early diagnosis of biliary tract cancer (BTC) is important for curative surgical resection. Current tumor markers of BTC are unsatisfactory in terms of sensitivity and specificity. In a search for novel biomarkers for BTC, serum samples obtained from 62 patients with BTC were compared with those from patients with benign biliary diseases and from healthy controls, using the MALDI-TOF/TOF ClinProt system. Initial screening and further validation identified a peak at 4204 Da with significantly greater intensity in the BTC samples. The 4204 Da peak was partially purified and identified as a fragment of prothrombin by amino acid sequencing. The sensitivity of the 4204 Da peptide for detection of stage I BTC cancer was greater than those for CEA and CA19-9. Also, serum levels of the 4204 Da peptide were above the cut-off level in 15 (79%) of 19 cases in which the CEA and CA19-9 levels were both within their cut-off values. Receiver operating characteristic analysis showed that the combination of the 4204 Da peptide and CA19-9 was significantly more sensitive for detection of stage I BTC cancer compared to CEA and CA19-9. These results suggest that this protein fragment may be a promising biomarker for biliary tract cancer.
International journal of proteomics. 01/2012; 2012:108609.
[show abstract][hide abstract] ABSTRACT: The representative tumor markers for HCC, AFP, and PIVKA-II are not satisfactory in terms of sensitivity and specificity in the early diagnosis of HCC. In search for novel markers for HCC, three-step proteome analyses were carried out in serum samples obtained from 12 patients with HCC and 10 with LC. As a first step, serum samples were subjected to antibody-based immunoaffinity column system that simultaneously removes twelve of abundant serum proteins. The concentrated flow-through was then fractionated using reversed-phase HPLC. Proteins obtained in each fraction were separated by SDS-PAGE. Serum samples obtained from patient with HCC and with LC were analyzed in parallel and their protein expression patterns were compared. A total of 83 protein bands were found to be upregulated in HCC serum. All the protein bands, the intensity of which was different between HCC and LC groups, were identified. Among them, clusterin was most significantly overexpressed (P = 0.023). The overexpression of serum clusterin was confirmed by ELISA using another validation set of HCC samples. Furthermore, serum clusterin was elevated in 40% of HCC cases in which both AFP and PIVKA-II were within their cut-off values. These results suggested that clusterin is a potential novel serum marker for HCC.
International journal of proteomics. 01/2012; 2012:623190.
[show abstract][hide abstract] ABSTRACT: Serum biomarkers currently available for gastric cancers are not sufficiently sensitive and specific.
We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to generate comparative peptide profiles of serum samples obtained from gastric cancer patients (n = 81) and age- and sex-matched healthy controls (n = 66).
Because of initial screening and further validation, we found that the intensities of a 2209 m/z MS peak were increased in the preoperative sera obtained from gastric cancer patients, and we identified this peak, a 2209 Da peptide, as a high molecular weight (HMW) kininogen fragment. Receiver operating characteristic analyses showed that the area under the curve (AUC) for the 2209 Da peptide (AUC = 0.715) was greater than those for conventional tumor markers (carcinoembryonic antigen AUC = 0.593, carbohydrate antigen 19-9 AUC = 0.527) used for the detection of stage I gastric cancers. Inverse correlations were observed between the levels of intact HMW kininogen and the 2209 Da peptide, suggesting that the upregulation of some protease activities is responsible for the overproduction of a kininogen fragment in gastric cancer patients.
Serum levels of the 2209 Da peptide identified in this study have a greater diagnostic ability than those of conventional tumor markers used for the early detection of gastric cancer.
Journal of Gastroenterology 02/2011; 46(5):577-85. · 3.79 Impact Factor
[show abstract][hide abstract] ABSTRACT: Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), causes the 78-kDa glucose-regulated protein (GRP78/BiP) cleavage, followed by induction of endoplasmic reticulum (ER) stress, leading to caspase-dependent apoptosis via mitochondrial membrane damage by Bax/Bak activation. The purpose of the present study was to identify SubAB receptors responsible for HeLa cell death. Four proteins, NG2, α2β1 integrin (ITG), L1 cell adhesion molecule (L1CAM), and hepatocyte growth factor receptor (Met), were identified to be SubAB-binding proteins by immunoprecipitation and purification, followed by liquid chromatography-tandem mass spectrometry analysis. SubAB-induced Bax conformational change, Bax/Bak complex formation, caspase activation, and cell death were decreased in β1 ITG, NG2, and L1CAM small interfering RNA-transfected cells, but unexpectedly, BiP cleavage was still observed. Pretreatment of cells with a function-blocking β1 ITG antibody (monoclonal antibody [MAb] P5D2) enhanced SubAB-induced caspase activation; MAb P5D2 alone had no effect on caspase activation. Furthermore, we found that SubAB induced focal adhesion kinase fragmentation, which was mediated by a proteasome-dependent pathway, and caspase activation was suppressed in the presence of proteasome inhibitor. Thus, β1 ITG serves as a SubAB-binding protein and may interact with SubAB-signaling pathways, leading to cell death. Our results raise the possibility that although BiP cleavage is necessary for SubAB-induced apoptotic cell death, signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways.
Infection and immunity 02/2011; 79(2):617-27. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: One possible factor determining recovery of trace amount of protein biomarker candidates during proteome analyses could be adsorption on urine tubes. This issue, however, has not been well addressed so far. Recently, a new technical device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (poly(MPC-co-BMA)) has been developed mainly to prevent the adsorption of plasma proteins. We assessed whether conventionally used urine tubes adsorb trace amount of urinary proteins and, if any, whether the surface coating by poly(MPC-co-BMA) can minimize the adsorption. Proteinuric urine samples were kept in poly(MPC-co-BMA)-coated and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or peptides onto urine tubes were analyzed by SDS-PAGE, 2-DE, and the MALDI-TOF MS. It was found that a number of proteins and/or peptides adsorb on the conventionally used urine tubes and that surface coating by poly(MPC-co-BMA) can minimize the adsorption without any significant effects on routine urinalysis test results. Although it remains to be clarified to what extent the protein adsorption can modify the results of urinary proteome analyses, one has to consider this possible adsorption of urinary proteins when searching for trace amounts of protein biomarkers in urine.
International journal of proteomics. 01/2011; 2011:502845.
[show abstract][hide abstract] ABSTRACT: We searched for novel tumor markers of pancreatic cancer by three-step serum proteome analysis. Twelve serum abundant proteins were depleted using immunoaffinity columns followed by fractionation by reverse-phase high-performance liquid chromatography. Proteins in each fraction were separated by two-dimensional gel electrophoresis. Then the gel was stained by Coomassie Brilliant Blue. Protein spots in which the expression levels were significantly different between cancer and normal control were identified by LC-MS/MS. One hundred and two spots were upregulated, and 84 spots were downregulated in serum samples obtained from patients with pancreatic cancers, and 58 proteins were identified by mass spectrometry. These candidate proteins were validated using western blot analysis and enzyme-linked immunosorbent assay (ELISA). As a result of these validation process, we could confirm that the serum levels of apolipoprotein A-IV, vitamin D-binding protein, plasma retinol-binding protein 4, and tetranectin were significantly decreased in patients with pancreatic cancer.
International journal of proteomics. 01/2011; 2011:628787.
[show abstract][hide abstract] ABSTRACT: The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip(®) system and the ClinProt™ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins.
Two serum samples-one on admission and one after 8 weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA).
Three-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 μg/ml) than in healthy subjects without a drinking history (5.5 ± 3.0 μg/ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects.
Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.
Alcoholism Clinical and Experimental Research 11/2010; 35(2):211-7. · 3.42 Impact Factor