Han-Min Chen

AMPK activation inhibits expression of proinflammatory mediators through downregulation of PI3K/p38 MAPK and NF-κB signaling in murine macrophages.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in energy homeostasis and regulation of inflammatory responses. The present study is aimed to investigate the anti-inflammatory effects of ENERGI-F704, a nucleobase analogue isolated from bamboo leaves, on expression of proinflammatory mediators in murine macrophage RAW264.7 in response to lipopolysaccharide (LPS). ENERGI-F704 enhanced phosphorylation of AMPK(T172) but insignificantly affected the viability of RAW264.7 cells. Further investigation showed that ENERGI-F704 decreased mRNA expression of interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) induced by LPS, as well as suppressed the production of prostaglandin E2 (PGE₂) and nitric oxide (NO). Additionally, the inhibitory effects of ENERGI-F704 on the LPS-induced proinflammatory mediators were diminished by pretreatment of AMPK inhibitor Compound C. ENERGI-F704 also inhibited LPS-triggered activation of nuclear factor kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38), whereas extracellular signal-regulated kinase (Erk)1/2 and c-Jun N-terminal kinase (JNK) were insignificantly influenced. Our findings indicate that ENERGI-F704 may exert anti-inflammatory activity on RAW264.7 cells in response to LPS through the activation of AMPK and suppression of PI3K/P38/NF-κB signaling and the consequent decreased expression of proinflammatory mediators, suggesting that ENERGI-F704 is beneficial to the amelioration of inflammatory disorders.

A comprehensive evaluation of imidazole-zinc reverse stain for current proteomic researches

In this paper, we comprehensively evaluated the capability of imidazole‐zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2‐D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two‐third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.

Amelioration of LPS-induced inflammation response in microglia by AMPK activation.

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis via modulating metabolism of glucose, lipid, and protein. In addition to energy modulation, AMPK has been demonstrated to associate with several important cellular events including inflammation. The results showed that ENERGI-F704 identified from bamboo shoot extract was nontoxic in concentrations up to 80 μM and dose-dependently induced phosphorylation of AMPK (Thr-172) in microglia BV2 cells. Our findings also showed that the treatment of BV2 with ENERGI-F704 ameliorated the LPS-induced elevation of IL-6 and TNF-α production. In addition, ENERGI-F704 reduced increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) via downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively. Moreover, ENERGI-F704 decreased activated nuclear translocation and protein level of NF-κB. Inhibition of AMPK with compound C restored decreased NF-κB translocation by ENERGI-F704. In conclusion, ENERGI-F704 exerts inhibitory activity on LPS-induced inflammation through manipulating AMPK signaling and exhibits a potential therapeutic agent for neuroinflammatory disease.

Investigation of the effect of hyperglycemia on intracerebral hemorrhage by proteomic approaches

Intracerebral hemorrhage (ICH) is associated with high mortality and disability, and hyperglycemia worsens the clinical and neurological outcomes of patients with ICH. In this study, we utilized proteomic approaches to investigate the role of hyperglycemia in ICH. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ) in adult Sprague–Dawley male rats; ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. It was observed that the size of induced hemorrhage was significantly larger in the hyperglycemic group (n=6 in each group). On the first day after ICH, an apparent decrease in the bilateral grasp was also observed for the lesioned hyperglycemic rats compared with normoglycemic ones. When employing 2‐DE and MS to examine the proteomes of perihematomal and control regions in individual hyperglycemic and normoglycemic rats, eight differentially expressed protein targets were identified. Most noteworthy, in response to ICH significant increase of albumin was ubiquitously observed in the brains of normoglycemic rats but not in the brains of hyperglycemic rats. Coincidentally, more significant neuronal apoptosis were found in the perihematomal regions of hyperglycemic rats. These observations described suggest the protection role of albumin in acute stage of ICH, which may be dependent on different blood sugar levels.

Non-proteolytic house dust mite allergen, Der p 2, upregulated expression of tight junction molecule claudin-2 associated with Akt/GSK-3β/β-catenin signaling pathway.

Non‐proteolytic group 2 allergen, Der p 2 (DP2) is known as a major allergen derived from house dust mite Dermatophagoides pteronyssinus. Paracellular epithelial barrier, being composed of a number of tight junction (TJ) molecules, plays pivotal roles in resistance of pathogen invading. However, whether DP2 affects epithelial TJ molecules is unclear. Therefore, we aimed to investigate the effects of DP2 on epithelial TJ molecules, and the mechanism by which expression of junction molecules is regulated by DP2. Cell cycle and mRNA expression of TJ proteins of lung alveolar cell A549 were analyzed by RT‐PCR and flow cytometry. Level of claudin‐2, subcellular distribution of β‐catenin and kinase activation was determined using immunoblot. Our findings revealed that DP2 had no significant influence on cell cycle distribution but affected mRNA expression of TJ molecules including claudin‐2, occludin, and ZO‐1 in A549 cells. Our results showed that DP2 significantly elevated level of claudin‐2 and increased expression and nuclear translocation of β‐catenin. Moreover, DP2 enhanced the phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) and its potential upstream regulator Akt. The DP2‐induced claudin‐2 expression was also suppressed by GSK‐3β inhibitor (lithium chloride) and phosphatidyl inositol 3‐phosphate kinase (PI3K) inhibitor (wortamannin). Taken together, these findings showed that DP2 increased claudin‐2 expression and its cell surface distribution in A549 cells, which may attribute to phosphorylation of GSK‐3β and Akt and the consequent increase and nuclear translocation of β‐catenin. It is suggested that presence of DP2 may alter epithelial junction by regulating expression of TJ molecules. J. Cell. Biochem. 112: 1544–1551, 2011.

Ocimum gratissimum Aqueous Extract Induces Apoptotic Signalling in Lung Adenocarcinoma Cell A549

Ocimum gratissimum (OG) is widely used as a traditional herb for its antibacterial activity in Taiwan. Recently, antitumor effect of OG on breast cancer cell is also reported; however, the effects of OG on human pulmonary adenocarcinoma cell A549 remain unclear. Therefore, we aimed to investigate whether aqueous OG extract (OGE) affects viability of A549 cells and the signals induced by OGE in A549 cells. Cell viability assays revealed that OGE significantly and dose-dependently decreased the viability of A549 cell but not that of BEAS-2B cell. Morphological examination and DAPI staining indicated that OGE induced cell shrinkage and DNA condensation for A549 cells. Further investigation showed that OGE enhanced activation of caspase-3, caspase-9 and caspase-8 and increased protein level of Apaf-1 and Bak, but diminished the level of Bcl-2. Additionally, OGE inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) yet enhanced the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38). In conclusion, our findings indicate that OGE suppressed the cell viability of A549 cells, which may result from the activation of apoptotic signaling and the inhibition of anti-apoptotic signaling, suggesting that OGE might be beneficial to lung carcinoma treatment.

Amyloid P component as a plasma marker for Parkinson's disease identified by a proteomic approach

OBJECTIVES Parkinsons disease (PD) ranks the second among the neurodegenerative disorders. Proteins involved in Parkinsons disease (PD) have been investigated but none as the diagnostic markers in blood. DESIGN AND METHODS In this study, we applied a proteomic strategy, by utilizing two-dimensional electrophoresis and mass spectrometry, to analyze two sample pools of plasma from the healthy individuals and PD subjects. RESULTS IgGκL and human serum amyloid P component (SAP) were found differentially expressed between these pools. SAP level increased by approximately 5-fold in PD samples, and the ELISA procedure revealed a significant (P<0.001) increase in SAP concentration (65.9 ± 18.7μg/mL) in the plasma of PD subjects (healthy individuals, 35.0 ± 12.5μg/mL), with sensitivity of 94.1% and specificity of 87.5%. CONCLUSION Our results indicated a potential feasibility of plasma SAP as a marker to approach PD.

Plastidial Starch Phosphorylase in Sweet Potato Roots Is Proteolytically Modified by Protein-Protein Interaction with the 20S Proteasome

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions.

IL‐6 augmented motility of airway epithelial cell BEAS‐2B via Akt/GSK‐3β signaling pathway

Cell migration plays a pivotal role in airway repair and remodeling involved in respiratory diseases such as asthma. Interleukin‐6 (IL‐6) and fascin‐1 are involved in cell migration upon stimulation; however, the roles of IL‐6 and fascin‐1 in migration of airway epithelial cell remain sketchy. The present study was aimed to investigate influence of IL‐6 on cell motility with emphasis on the association with fascin‐1. Wound healing assay and transmigration assay were performed to examine effect of IL‐6 on migration and invasiveness of human bronchial epithelial cell BEAS‐2B. Level of mRNA expression was determined by RT‐PCR and quantitative real‐time RT‐PCR (Q‐PCR). Involvement of kinase and transcription factor signaling in IL‐6‐induced cell migration was investigated using immunoblot and specific inhibitors. IL‐6 significantly augmented cell migration and invasiveness in parallel with elevated fascin‐1 expression. Further investigation showed that IL‐6 dose‐dependently upregulated fascin‐1 expression in both mRNA and protein levels. We showed that IL‐6 activated Akt and inhibited glycogen synthase kinase‐3β (GSK‐3β), highly associating with fascin‐1 mRNA expression. Additionally, IL‐6‐induced migration was significantly diminished by phosphatidyl inositol 3‐phosphate kinase (PI3K) inhibitor (wortamannin) and β‐catenin inhibitor FH535. Moreover, LiCl and SB216763, inhibitors of GSK‐3β augmented cell migration as well as fascin‐1 mRNA expression. Conclusively, these findings reveal that IL‐6‐induced migration of BEAS‐2B cell may be attributed to activation of Akt, inhibition of GSK‐3β, and the associated increase of β‐catenin and fascin‐1 expression, indicating an important role of Akt/GSK‐3β signaling and β‐catenin/fascin‐1 in IL‐6 associated airway remodeling. J. Cell. Biochem. 113: 3567–3575, 2012.

Use of backlit light plate to enhance visualization of imidazole-zinc reverse stained gels.

Imidazole-zinc reverse stain utilizing imidazole and zinc ions for protein visualization on electrophoresis gels was originally introduced in the 1990s (1–3). This method is based on the selective precipitation of imidazolate-zinc complex in the gel (3), except zones where proteins or other macromolecules, such as DNA (4–7) or lipopolysaccharide, are present (4,5,8,9). There are several advantages of using imidazole-zinc reverse stain in current proteomic research. First, imidazole-zinc reverse stain is highly sensitive. For protein gels, zinc reverse stain has been demonstrated to provide an equal or better staining sensitivity than Sypro Ruby stain or some silver stains (4,5). Protein, as low as 1 ng, may be detected in the gel by the reverse stain. Second, performing imidazolezinc reverse stain is exceedingly simple as compared with silver stain, which requires tedious preparation of fresh staining solutions. Imidazole-zinc reverse stain uses only two staining solutions, which can be easily diluted from stock solutions. Third, it is remarkably fast, generally taking less than 20 min to complete (4,5,10). Finally, imidazole-zinc reverse stain is fully compatible to down-stream applications, such as mass spectrometry (MS) (4,11–14), Edman sequencing (1–3), electroelution (1,2,4,6), and membrane blotting techniques (4,5). Although the dynamic range of staining for imidazole-zinc reverse stain might not be as satisfactory as for Sypro Rube stain, nowadays laboratories that are not equipped with synchronized spot pickers on their fluorescent scanners sometimes use it to restain the Sypro Ruby stained two-dimensional electrophoresis (2-DE) gels, then manually pick the interested protein spots for identification by MS. The imidazole-zinc reverse stained gel delivers an image with transparent bands, spots, and a white background. When the gel is placed above a dark Use of backlit light plate to enhance visualization of imidazole-zinc reverse stained gels