[show abstract][hide abstract] ABSTRACT: Objective: Previous histochemical studies in demyelinating form of Guillain-Barré
syndrome (GBS), acute inflammatory demyelinating polyneuropathy (AIDP), have shown
complement deposition on the surface of the Schwann cells, and therefore unknown
epitopes would be present on the outer surface of the Schwann cells.
Methods: We used proteomic-based approach to search for the target molecules of AIDP
in the extracted proteins from schwannoma cells. Sera were obtained from 40 GBS patients,
31 inflammatory disease controls, and 46 normal controls.
Results: We found that patients with AIDP after cytomegalovirus (CMV) infection have
serum autoantibodies against membrane-organizing extension spike protein (moesin),
which is expressed in the Schwann cell processes at the nodes of Ranvier and is crucial for
myelination. Of the 40 GBS patients, six had recent CMV infection and five of them (83%)
had high levels of serum IgG antibodies against moesin. The anti-moesin antibodies were
found in none of disease controls including five with CMV infection but no neuropathy, and
only two (4%) of the 46 normal control subjects. Immunocytochemistry showed that moesin
was stained at the distal tips of schwannoma cells by sera from the CMV-related AIDP
patients but not by sera from controls.
Conclusion: Moesin is a possible immunological target molecule of pathogenic
autoantibodies in patients with CMV-related AIDP.
Classification of Evidence: This study provides Class II evidence that levels of serum
anti-moesin antibodies accurately distinguishes CMV-related AIDP from non-CMV related
AIDP (sensitivity 83%, specificity 93%).
[show abstract][hide abstract] ABSTRACT: The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation, and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer (HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside of the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its down-regulation causes CIN in colorectal cancer.
[show abstract][hide abstract] ABSTRACT: Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer's disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.
Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.
The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.
Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.
PLoS ONE 01/2014; 9(1):e85356. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Objectives: Recent advances in sophisticated technologies in proteomics should provide promising ways to discover novel markers for hepatocellular carcinoma (HCC) in the early diagnosis. Methods: Serum peptide and protein profiling was conducted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Profiling was carried out in a training set of 16 patients with HCC and a testing set of 15 patients with cirrhosis without HCC. All the patients were hepatitis C virus positive. Candidate peaks were processed to partial purification, followed by protein identification by amino acid sequence analysis. Immunoprecipitation was conducted to confirm the protein identity. Results: Partial purification and protein identification revealed that one peak that was up-regulated in HCC sera both in the training and the testing sets was a fragment of apolipoprotein A-I (apo A-I). Immunoprecipitation confirmed this result. Conclusions: MALDI-TOF MS analysis revealed that apo A-I is a potential novel serum marker of HCC. Combination of these pretreatments and the current magnet bead-assisted MALDI-TOF MS will further enhance the efficiency of biomarker discovery for HCC.
American Journal of Clinical Pathology 01/2014; 141(1):52-61. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. In recent years, studies have shown a definite association between periodontal disease and other inflammatory conditions of the body. High-throughput analysis of proteins has become possible with the development of mass spectrometry technology. This breakthrough in proteome technology enables comparative studies of comprehensive protein expression and identification of protein. In case of periodontal disease, proteome analysis using two-dimensional electrophoresis (2-DE), as well as gel-free methods, has been reported. As a fluid lying in close proximity to periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for biomarkers of periodontal disease, because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Increasing numbers of recent reports have provided evidence that the proteomic approach is a promising tool for the discovery and identification of biochemical markers of periodontal disease. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize recent comprehensive proteomic studies aimed at discovering and identifying biomarkers of periodontal disease in GCF.
[show abstract][hide abstract] ABSTRACT: Schizophyllum commune is one of the causative agents of basidiomycosis including disorders such as allergic bronchopulmonary mycosis, allergic fungal sinusitis, and mucoid impaction of bronchi, the incidence of those of which has been increasing. These mycoses are difficult to diagnose because only a limited number of diagnostic tools are currently available. The biggest problem is that no specific antigens of S. commune have been identified to enable serodiagnosis of the disease.
In this study, we attempted to identify a major antigen of S. commune to establish a reliable serodiagnostic method.
We used mass spectrometry to identify an antigen that reacted with the serum of a patient with allergic bronchopulmonary mycosis caused by S. commune. The protein was expressed in Escherichia coli, highly purified, and the patient sera IgG and IgE titers against the protein were determined by enzyme-linked immunosorbent assay.
The protein identified as a major antigen of S. commune was named Sch c 1; it was a homolog of glucoamylase. The IgG and IgE titers against Sch c 1 in patient sera were significantly higher than those in healthy volunteer sera (p < 0.01).
Sch c 1 is recognized by the host immune system of patients as an antigen/allergen. The purified glucoamylase Sch c 1 is a promising candidate antigen for the serodiagnosis of S. commune-induced mycosis. This article is protected by copyright. All rights reserved.
[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.
[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