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Kazuyasu Fujii

Publications and source records attributed to Kazuyasu Fujii.

13 recordsLinked to original sources
PubMed2006

Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye.

CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.

Cell Line, Tumor↗
PubMed2006

Toward a comprehensive quantitative proteome database: protein expression map of lymphoid neoplasms by 2-D DIGE and MS.

Using 2-D DIGE, we constructed a quantitative 2-D database including 309 proteins corresponding to 389 protein spots across 42 lymphoid neoplasm cell lines. The proteins separated by 2-D PAGE were identified by MS and assigned to the expression data obtained by 2-D DIGE. The cell lines were categorized into four groups: those from Hodgkin's lymphoma (HL) (4 cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell malignancies (3 cell lines). We characterized the proteins in the database by classifying them according to their expression level. We found 28 proteins with more than a 2-fold difference between the cell line groups. We also noted the proteins that allowed multidimensional separation to be achieved (1) between HL cells and other cells, (2) between the cells derived from B cells, T cells and NK cells, and (3) between HL cells and anaplastic large cell lymphoma cells. Decision tree classification identified five proteins that could be used to classify the 42 cell lines according to differentiation. These results suggest that the quantitative 2-D database using 2-D DIGE will be a useful resource for studying the mechanisms underlying the differentiation phenotypes of lymphoid neoplasms.

Algorithms↗
PubMed2005

Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye.

To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.

Adult↗
PubMed2005

Proteomic signatures for histological types of lung cancer.

We performed proteomic studies on lung cancer cells to elucidate the mechanisms that determine histological phenotype. Thirty lung cancer cell lines with three different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma and adenocarcinoma) were subjected to two-dimensional difference gel electrophoresis (2-D DIGE) and grouped by multivariate analyses on the basis of their protein expression profiles. 2-D DIGE achieves more accurate quantification of protein expression by using highly sensitive fluorescence dyes to label the cysteine residues of proteins prior to two-dimensional polyacrylamide gel electrophoresis. We found that hierarchical clustering analysis and principal component analysis divided the cell lines according to their original histology. Spot ranking analysis using a support vector machine algorithm and unsupervised classification methods identified 32 protein spots essential for the classification. The proteins corresponding to the spots were identified by mass spectrometry. Next, lung cancer cells isolated from tumor tissue by laser microdissection were classified on the basis of the expression pattern of these 32 protein spots. Based on the expression profile of the 32 spots, the isolated cancer cells were categorized into three histological groups: the squamous cell carcinoma group, the adenocarcinoma group, and a group of carcinomas with other histological types. In conclusion, our results demonstrate the utility of quantitative proteomic analysis for molecular diagnosis and classification of lung cancer cells.

Adenocarcinoma↗
PubMed2005

Protein expression pattern distinguishes different lymphoid neoplasms.

To identify proteins associated with the histological subtypes of lymphoid neoplasms, we studied the proteomes of 42 cell lines from human lymphoid neoplasms including Hodgkin's lymphoma (HL; four cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell lymphoma (three cell lines). The protein spots were sequentially selected by (i) Wilcoxon or Kruskal-Wallis tests to find the spots whose intensity was significantly (p <0.05) different among the cell line groups, (ii) by statistical-learning methods to prioritize the spots according to their contribution to the classification, and (iii) by unsupervised classification methods to validate the classification robustness by the selected spots. The selected spots discriminated (i) between HL cells and other cells, (ii) between the cells from B cell malignancies, T cell malignancies, and NK cell lymphoma cells, and (iii) between HL cells and anaplastic large cell lymphoma cells. Among the 31 informative protein spots, MS identified 24 proteins corresponding to 23 spots. Previous reports did not correlate these proteins to lymphocyte differentiation, suggesting that a proteomic study would identify the novel mechanisms responsible for the histogenesis of lymphoid neoplasms. These proteins may have potential as differential diagnostic markers for lymphoid neoplasms.

Amino Acid Sequence↗
PubMed2005

Effect of formaldehyde gas exposure in a murine allergic contact hypersensitivity model.

To clarify the effect of formaldehyde (FA) gas exposure on contact hypersensitivity (CHS), CHS reactions against 2,4,6-trinitrochlorobenzene (TNCB) was studied in BALB/c mice with a low dose of FA gas exposure. The TNCB-induced CHS reactions were slightly suppressed by the FA gas exposure immediately after sensitization, whereas they were significantly enhanced and prolonged in mice continuously exposed to FA gas before and after sensitization. We showed that exposure to FA gas enhanced the Th2 dominant responses in draining lymph node (LN) in early stage of CHS. In contrast, T cell subsets and their intracellular cytokine production in the draining LN were similar during the early stage of CHS by FA gas exposure during the sensitization phase. The percentage of CD8+ T cells was increased, and the percentage of CD4+CD25+ T cells was decreased in the FA gas-exposed group at 72 hr after elicitation. These results indicate that FA gas-exposed might influence regulatory T cells. Furthermore, in the chronic CHS model that was repetitively elicited with TNCB, more intensive and prolonged CHS reactions, and increased numbers of mast cells were found in the FA gas-exposed group at 4 hr after elicitation than in the control group, FA gas exposure may alter the intensity of allergic CHS.

Administration, Cutaneous↗
PubMed2004

Proteomic signature corresponding to alpha fetoprotein expression in liver cancer cells.

Alpha fetoprotein (AFP) has been implicated in the development of hepatocellular carcinoma and is considered to be a diagnostic and prognostic tumor marker. Because elevated expression of AFP is associated with many characteristics of hepatocellular carcinoma tissues, we hypothesized that multiple proteins may function in a coordinated manner with AFP. To identify such proteins, we performed global protein expression analysis, namely a proteomic study. The protein expression profiles of 9 AFP-producing liver cancer cell lines (JHH-5, HuH-1, PLC/PRL/5, Hep3B, HT-17, JHH-7, HuH-7, HepG2, Li-7) and 7 nonproducing liver cancer cell lines (HLE, JHH-6, Sk-Hep-1, JHH-4, HLF, RBE, SSP-25) were generated by fluorescence 2-dimensional difference gel electrophoresis. In fluorescence 2-dimensional difference gel electrophoresis, proteins are labeled with fluorescent dyes before electrophoresis for more accurate quantitative expression analysis. We identified 11 protein spots that distinguished AFP-producing cell lines from nonproducing cell lines by multivariate studies. The spots showed consistent alterations in amount in AFP-producing cell lines (6 up-regulated and 5 down-regulated). An additional 5 liver cancer cell lines (KIM-1, KYN-2, KYN-3, PH5-CH, PH5-T) also were correctly grouped with respect to their AFP production on the basis of the intensity of the 11 protein spots. The proteins corresponding to the 11 selected spots were identified by mass spectrometry and were categorized into 4 groups based on their known role in apoptosis, glucose metabolism, cytoskeletal organization, or translation. In conclusion, we found a novel association of AFP with other proteins. Their interaction should provide insight into the biology of AFP-producing hepatocellular carcinoma cells.

Cell Line, Tumor↗
PubMed2004

Proteomic signature of human cancer cells.

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.

Cell Line, Tumor↗
PubMed2004

A spectrum of clinical manifestations caused by host immune responses against Epstein-Barr virus infections.

Epstein-Barr virus (EBV), or human herpesvirus 4 (HHV-4), infects the vast majority of adults worldwide, and establishes both nonproductive (latent) and productive (lytic) infections. Host immune responses directed against both the lytic and latent cycle-associated EBV antigens induce a diversity of clinical symptoms in patients with chronic active EBV infections who usually contain an oligoclonal pool of EBV-infected lymphocyte subsets in their blood. Episomal EBV genes in the latent infection utilize an array of evasion strategies from host immune responses: the minimized expression of EBV antigens targeted by host cytotoxic T lymphocytes (CTLs), the down-regulation of cell adhesion molecule expression, and the release of virokines to inhibit the host CTLs. The oncogenic role of latent EBV infection is not yet fully understood, but latent membrane proteins (LMPs) expressed during the latency cycle have essential biological properties leading to cellular gene expression and immortalization, and EBV-encoded gene products such as viral interleukin-10 (vIL-10) and bcl-2 homologue function to survive the EBV-infected cells. The subsequent oncogenic DNA damage may lead to the development of neoplasms. EBV-associated NK/T cell lymphoproliferative disorders are prevalent in Asia, but quite rare in Western countries. The genetic immunological background, therefore, is closely linked to the development of EBV-associated neoplasms.

Epstein-Barr Virus Infections↗
PubMed2003

Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool.

The combination of laser microdissection and two-dimensional gel electrophoresis (2-D PAGE) has been developed to perform proteomic analysis on specific populations of cells in cancer tissues. However, as conventional low sensitivity silver staining was used for spot detection, the microdissection required to obtain an adequate amount of protein for 2-D PAGE is laborious and only a restricted number of protein spots could be visualized. As a consequence, this technology was impractical for direct clinical applications and had a limited impact on cancer studies. To solve these problems, we developed an application in which fluorescent dyes label the proteins extracted from microdissected tissues prior to 2-D PAGE separation. In this application, a small amount of protein, less than 6.6 microg, was enough to generate a 2-D profile with approximately 1500 protein spots. This technique was applied to compare the proteome of normal intestinal epithelium with that of adenoma in Min mice. Thirty-seven protein spots reproducibly showed significant differences in intensities. Mass spectrometric analysis and Western blotting identified eight of them, including prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84. These results indicate that fluorescence labeling of proteins from microdissected tissues prior to 2-D PAGE is a powerful cancer proteomic study tool.

Adenoma↗
PubMed2002

Confocal laser microscopic observation of glycocalyx production by Staphylococcus aureus in vitro.

We used a scanning confocal laser microscope to study the effects of various agents on sugar production by Staphylococcus aureus in vitro. S. aureus cells attached to coverslips in Pl-TSB (plasma:tryptic soy broth=1:1) were stained with fluorescein isothiocyanate-conjugated concanavalin A (FITC-conA) and were more strongly stained over time. We considered that the materials that stained positive for FITC-conA consistent with S. aureus cells were sugars, probably glycocalyx, produced by the S. aureus cells. Since the cells in the stationary growth phase alone were strongly stained with FITC-conA, all S. aureus cells attached to the coverslips in Pl-TSB were considered to be in this phase (low growth rate). The positive staining for FITC-conA was markedly reduced when fibrin was not formed in Pl-TSB with plasmin and sucrose, and was also markedly reduced when the fibrin in Pl-TSB was destroyed with plasmin. In conclusion, the results of the present study indicate that the existence of fibrin is essential for glycocalyx production and biofilm formation of S. aureus cells to aid in the attachment of S. aureus cells in vitro, because S. aureus cells attached on coverslips and fibrin alone produce glycocalyx. Of the antimicrobial agents tested, sulfadiazine silver most strongly inhibited the production of FITC-conA-positive materials by S. aureus cells at a sub-MIC concentration. Plasmin, sucrose, and sulfadiazine silver may be useful topical applications for use on clinical dermatology for the prevention and the treatment of staphylococcal biofilms. We consider that this simple method is very useful for the detection of S. aureus glycocalyx on dermatology field.

Biofilms↗
PubMed2002

Initial recruitment of interferon-gamma-producing CD8+ effector cells, followed by infiltration of CD4+ cells in 2,4,6-trinitro-1-chlorobenzene (TNCB)-induced murine contact hypersensitivity reactions.

Contact hypersensitivity (CHS) is an antigen-specific, T-cell-mediated skin reaction in sensitized individuals. Recent studies have demonstrated that the murine CHS reaction to 2,4-dinitrofluorobenzene (DNCB) is mediated by CD8+ T cells and down-regulated by CD4+ T cells. We studied cellular events and functions of infiltrating cells in CHS reactions to 2,4,6-trinitro-1-chlorobenzene (TNCB) in the skin and draining lymph nodes (LN) of BALB/c mice and compared them with the CHS reaction to a house dust mite antigen, Dermatophagoides farinae (Df). Mice were sensitized with TNCB or Df antigens, and CHS reactions elicited with the corresponding antigens were examined immunohistologically. Cytokines produced by individual cells isolated from the CHS reactions were analyzed by flow cytometry, and the expression of cytokine mRNA was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Results demonstrated that the intensity of TNCB-elicited CHS skin reactions reached its peak at 24 hr after elicitation and decreased gradually. Flow cytometric analysis of isolated cells from the TNCB-elicited CHS reactions demonstrated that the infiltrating cells were composed of approximately 25% CD4+ and CD8+ cells at 12, 24, and 36 hr after challenge, although infiltrating cells became dense at 36 hr by histological observation. The percentage of IFN-gamma-producing CD8+ cells (Tc1) in the cell fractions reached its peak at 12 hr and decreased gradually. The peak infiltration of IFN-gamma-producing CD4+ cells (Th 1) was observed at 24 hr. IL-4-producing cells, however, were always below 5% in the cell fractions. The RT-PCR method demonstrated that IFN-gamma mRNA was detected in the TNCB-elicited skin reactions at 12, 18 and 24 hr after elicitation, became weak at 48 hr, and disappeared at 72 hr. No IL-4 mRNA was detected from 12 to 72 hr. In the draining LN cells, however, the percentages of both IFN-gamma-producing CD8+ (Tc1) and CD4+ cells (Th1) decreased 12 to 36 hr after TNCB elicitation. CHS reactions of Df antigens were predominantly composed of infiltrations of CD4+ cells in to the skin, associated with the expression of IL-4 mRNA from 12 to 48 hr after elicitaion. The expression of IFN-gamma mRNA was detected at 48 hr or later. Our findings indicate that the CHS skin reaction to TNCB is induced by early recruitment of IFN-gamma-producing CD8+ effector cells (Tcl), followed by infiltration of IFN-gamma-producing CD4+ cells (Th1), whereas IL-4-producing T cells (Th2) induce the early CHS response to Df antigens.

Animals↗