Shui-Tein Chen

Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities.

A fucose-containing glycoprotein fraction which stimulates spleen cell proliferation and cytokine expression has been identified from the water-soluble extract of Ganoderma lucidum. Proteomic analysis of mouse spleen cells treated with this glycoprotein fraction showed approximately 50% change of the proteome. Further studies on the activities of this glycoprotein fraction through selective proteolysis and glycosidic cleavage indicate that a fucose containing polysaccharide fraction is responsible for stimulating the expression of cytokines, especially IL-1, IL-2 and INF-gamma.

Purification and characterization of two cellulase free xylanases from an alkaliphilic Bacillus firmus

Two xylanases from Bacillus firmus were purified to homogeneity by gel filtration and ion-exchange chromatography. These enzymes have molecular weights of 45 kDa and 23 kDa, respectively, and both show enzymatic activity over the pH range of 5.0–11.0 at 37°C. These enzymes hydrolyzed xylan from birchwood to release mainly the products of xylose, xylotriose and xylohexose, thus indicating that the xylanases act preferentially toward the internal glycosidic bonds of xylo-oligosaccharides. However, the two xylanases show different modes of action, and a combination of both is likely to lead to concerted degradation of xylan down to the mono- and disaccharides.

Alterations in actin cytoskeletal assembly and junctional protein complexes in human endothelial cells induced by dengue virus infection and mimicry of leukocyte transendothelial migration.

Vascular leakage is a hallmark of severe dengue infection. Although extensive studies have been conducted during the past several decades, the molecular mechanisms underlying vascular leakage in dengue shock syndrome (DSS) remain unclear. We thus performed a proteomics study to characterize responses in human endothelial cells (EA.hy926) after DEN-2 virus infection (MOI=10). Comparative 2-D PAGE analysis revealed significantly altered abundance levels of 15 proteins, which were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS). These altered proteins were involved in several biological processes, for example, mRNA stability/processing, transcription and translation regulation, molecular chaperoning, oxidative stress response/regulation, cytoskeletal assembly, protein degradation, and cellular metabolisms. We also performed functional analyses of alterations in actin cytoskeletal assembly and endothelial integrity focusing on adherens junction (VE-cadherin), tight junction (ZO-1) and adhesive molecule (PECAM-1) after 24-h of DEN-2 infection and simulation of transendothelial migration by PECAM-1 cross-linking. Decreased expression and disorganization of the actin-cytoskeleton were observed in the infected cells, whereas the increase in actin stress fibers was found in adjacent noninfected cells. Additionally, a decrease in adhesive protein PECAM-1 was observed. Furthermore, DEN-2 infection caused decreased expression and redistribution of both VE-cadherin and ZO-1, whose changes were enhanced by PECAM-1 engagement. These alterations may potentially be a molecular basis explaining increased endothelial permeability or vascular leakage in DSS.

Coregulation of transcription factors and microRNAs in human transcriptional regulatory network

BackgroundMicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the post-transcriptional level. Recent studies have suggested that miRNAs and transcription factors are primary metazoan gene regulators; however, the crosstalk between them still remains unclear.MethodsWe proposed a novel model utilizing functional annotation information to identify significant coregulation between transcriptional and post-transcriptional layers. Based on this model, function-enriched coregulation relationships were discovered and combined into different kinds of functional coregulation networks.ResultsWe found that miRNAs may engage in a wider diversity of biological processes by coordinating with transcription factors, and this kind of cross-layer coregulation may have higher specificity than intra-layer coregulation. In addition, the coregulation networks reveal several types of network motifs, including feed-forward loops and massive upstream crosstalk. Finally, the expression patterns of these coregulation pairs in normal and tumour tissues were analyzed. Different coregulation types show unique expression correlation trends. More importantly, the disruption of coregulation may be associated with cancers.ConclusionOur findings elucidate the combinatorial and cooperative properties of transcription factors and miRNAs regulation, and we proposes that the coordinated regulation may play an important role in many biological processes.

GeneNetwork: an interactive tool for reconstruction of genetic networks using microarray data

UNLABELLED Inferring genetic network architecture from time series data generated from high-throughput experimental technologies, such as cDNA microarray, can help us to understand the system behavior of living organisms. We have developed an interactive tool, GeneNetwork, which provides four reverse engineering models and three data interpolation approaches to infer relationships between genes. GeneNetwork enables a user to readily reconstruct genetic networks based on microarray data without having intimate knowledge of the mathematical models. A simple graphical user interface enables rapid, intuitive mapping and analysis of the reconstructed network allowing biologists to explore gene relationships at the system level. AVAILABILITY Download from http://genenetwork.sbl.bc.sinica.edu.tw/. SUPPLEMENTARY INFORMATION Supplement documentation of algorithms for the four approaches is downloadable at the above location.

Proteomics reveals protein profile changes in doxorubicin - treated MCF-7 human breast cancer cells

MCF-7 cells are extensively used as a cell model to investigate human breast tumors and the cellular mechanism of antitumor drugs such as doxorubicin (DOX), an anthracycline antitumor drug widely used in clinical chemotherapy. To understand the effects of DOX on the protein expression, we perform a comprehensive proteomics to survey global changes in proteins after DOX treatment in MCF-7 cells. Exposure of MCF-7 cells to 0.1 microM DOX for 2 days induced a differentiation-like phenotype with prominent perinuclear autocatalytic vacuoles, abundant filamentous material, and irregular microvilli at the cell surface. In this study, we also present a proteome reference map of MCF-7 cells with 21 identified protein spots via analysis of N-terminal sequencing, mass spectrometry, immunoblot and/or computer matching with protein database. Based on the proteome map, we found that DOX causes a markedly decrease in the levels of three isoforms of heat shock protein 27 (HSP27) whereas the levels of other stress associated proteins including HSP60, calreticulin, and protein disulfide isomerase were not significantly altered in DOX-treated MCF-7 cells. Taken together, we suggest that that action of DOX on breast tumor cells may be partly related to dysregulation of HSP27 expression. Modulation of HSP27 levels may be a clinically useful potential target for design of antitumor drugs and controlling breast tumor growth.

The Ubiquitin−Proteasome Pathway Is Important for Dengue Virus Infection in Primary Human Endothelial Cells

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are the most severe forms of dengue virus infection with hemorrhage and plasma leakage. However, pathogenic mechanisms of DHF and DSS remain poorly understood. We therefore investigated host responses as determined by changes in the cellular proteome of primary human endothelial cells upon infection with dengue virus serotype 2 (DEN-2) at a multiplicity of infection (MOI) of 10 for 24 h. Two-dimensional PAGE and quantitative intensity analysis revealed 38 significantly altered protein spots (16 upregulated and 22 downregulated) in DEN-2-infected cells compared to mock controls. These altered proteins were successfully identified by mass spectrometry, including those involved in oxidative stress response, transcription and translation, cytoskeleton assembly, protein degradation, cell growth regulation, apoptosis, cellular metabolism, and antiviral response. The proteomic data were validated by Western blot analyses [upregulated ubiquitin-activating enzyme E1 (UBE1) and downregulated annexin A2] and an immunofluorescence study (upregulated MxA). Interestingly, we found that MxA was colocalized with DEN-2 viral capsid protein, strengthening its role as an antiviral protein. Moreover, we also identified upregulation of a proteasome subunit. Our functional study revealed the significant role of ubiquitination in dengue infection and UBE1 inhibition by its specific inhibitor (UBEI-41) caused a significant reduction in the level of viral protein synthesis and its infectivity. Our findings suggest that various biological processes were triggered in response to dengue infection, particularly antiviral IFN and ubiquitin-proteasome pathways.

Biomic study of human myeloid leukemia cells differentiation to macrophages using DNA array, proteomic, and bioinformatic analytical methods.

A biomic approach by integrating three independent methods, DNA microarray, proteomics and bioinformatics, is used to study the differentiation of human myeloid leukemia cell line HL‐60 into macrophages when induced by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Analysis of gene expression changes at the RNA level using cDNA against an array of 6033 human genes showed that 5950 (98.6%) of the genes were expressed in the HL‐60 cells. A total of 624 genes (10.5%) were found to be regulated during HL‐60 cell differentiation. Most of these genes have not been previously associated with HL‐60 cells and include genes encoded for secreted proteins as well as genes involved in cell adhesion, signaling transduction, and metabolism. Protein analysis using two‐dimensional gel electrophoresis showed a total of 682 distinct protein spots; 136 spots (19.9%) exhibited quantitative changes between HL‐60 control and macrophages. These differentially expressed proteins were identified by mass spectrometry. We developed a bioinformatics program, the Bulk Gene Search System (BGSS, http://www.sinica.edu.tw:8900/perl/genequery.pl) to search for the functions of genes and proteins identified by cDNA microarrays and proteomics. The identified regulated proteins and genes were classified into seven groups according to subcellular locations and functions. This powerful holistic biomic approach using cDNA microarray, proteomics coupled to bioinformatics can provide in‐depth information on the impact and importance of the regulated genes and proteins for HL‐60 differentiation.

Vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release

Our previous study using expression proteomics demonstrated that many proteins, particularly five forms of heterogeneous nuclear ribonucleoproteins (hnRNPs), were up-regulated in human endothelial cells upon dengue virus infection. To address functional significance of these proteins in response to dengue virus infection, we performed a functional proteomics study to identify hnRNPs-interacting proteins in the infected EA.hy926 cells. Immunoprecipitation followed by 2-D PAGE and mass spectrometric analyses revealed 18 and 13 interacting partners of hnRNP C1/C2 and hnRNP K, respectively. Interestingly, vimentin was a common partner for both hnRNP C1/C2 and K. The interaction between vimentin and these hnRNPs was confirmed by reciprocal immunoprecipitation followed by Western blot analysis and also by double immunofluorescence staining. Disruption of vimentin intermediate filament by acrylamide not only dissociated these complexes but also reduced nuclear hnRNPs expression, whereas cytosolic hnRNPs expression was unchanged. We also demonstrated that vimentin was strongly associated with dengue non-structural protein 1 (NS1). Disruption of vimentin intermediate filament not only dissociated this complex but also reduced dengue NS1 expression, as well as viral replication and release. Our data report for the first time that vimentin interacts with hnRNPs and dengue NS1, and plays a crucial role in replication and release of dengue virus.

Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression

We have previously reported that a well‐characterized glycoprotein fraction containing fucose residues in an extract of Ganoderma lucidum polysaccharides (EORP) exerts certain immuno‐modulation activity by stimulating the expression of inflammatory cytokines via TLR4. Continuing our studies, we have demonstrated that EORP increases the surface expression of CD14 and TLR4 within murine macrophages J774A.1 cells in vitro, and further promotes LPS binding and uptake by J774A.1 cells in a CD14‐dependent fashion. Moreover, we observed the co‐localization of internalized LPS with lysosome‐ and Golgi‐apparatus markers within 5 min after J774A.1 cells stimulated with LPS. In addition, EORP pretreatment of J774A.1 cells and human blood‐derived primary macrophages, followed by LPS stimulation, results in the super‐induction of interleukin‐1beta (IL‐1) expression. Endocytosis inhibitors: such as cytochalasin D and colchicine effectively block EORP‐enhanced LPS internalization by J774A.1 cells; yet they fail to decrease the LPS‐induced phosphorylation of certain mitogen‐activated protein kinases, and IL‐1 mRNA and proIL‐1 protein expression, indicating that LPS internalization by J774A.1 cells is not associated with LPS‐dependent activation. Our current results could provide a potential EORP‐associated protection mechanism for bacteria infection by enhancing IL‐1 expression and the clearance of contaminated LPS by macrophages. J. Cell. Physiol. 212: 537–550, 2007.