المؤلفون: Yi Li Huang, Wang Chou Sung, Yu Hua Lin, Sung Fang Chen, Ting Yu Wei, Sheng Yu Huang, Chiung Wen Chang, Han Min Chen

المصدر: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1864:1188-1194

مصطلحات موضوعية: 0301 basic medicine, Proteases, Stereochemistry, Disulfide Linkage, Thermolysin, Biophysics, Angiogenesis Inhibitors, 01 natural sciences, Biochemistry, Mass Spectrometry, Analytical Chemistry, 03 medical and health sciences, Hydrolysis, medicine, Trypsin, Amino Acid Sequence, Disulfides, Molecular Biology, Peptide sequence, chemistry.chemical_classification, Chemistry, 010401 analytical chemistry, Hydrogen-Ion Concentration, 0104 chemical sciences, Bevacizumab, Solutions, 030104 developmental biology, Enzyme, Biocatalysis, medicine.drug

الوصف: Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a26a386d515846a213c336f5dbcab09eTest
https://doi.org/10.1016/j.bbapap.2016.05.011Test

المؤلفون: Shang-Yi Chiang, Ming-Ling Tsai, Ann Chen, Hung-Te Liu, Pei-Hsiu Chen, Han-Min Chen, Yu-Ching Chou, Chih-Yuan Wang, Tzu-Hao Huang, Hao-Ai Shui, Yung-Fu Wu, Ching-Wu Hsia

المصدر: Journal of Proteomics. 75:2950-2959

مصطلحات موضوعية: Proteomics, Proteome, Angiogenesis, Biophysics, Validation Studies as Topic, Bioinformatics, Models, Biological, Biochemistry, Neuroprotection, Aqueous Humor, Western blot, medicine, Humans, Electrophoresis, Gel, Two-Dimensional, Eye Proteins, Aged, Aged, 80 and over, Diabetic Retinopathy, medicine.diagnostic_test, business.industry, Osmolar Concentration, Case-control study, Diabetic retinopathy, Middle Aged, medicine.disease, Pathophysiology, Case-Control Studies, business, Metabolic Networks and Pathways, Retinopathy

الوصف: Diabetic retinopathy (DR) can cause irreversible blindness and is the severest microvascular complication in the eyes of patients with diabetic mellitus (DM). The identification of susceptibility factors contributing to development of DR is helpful for identifying predisposed patients and improving treatment efficacy. Although proteomics analysis is useful for identifying protein markers related to diseases, it has never been used to explore DR-associated susceptibility factors in the aqueous humor (AH). To better understand the pathophysiology of DR and to identify DR-associated risk factors, a gel-based proteomics analysis was performed to compare AH protein profiles of DM patients with and without development of DR. MALDI-TOF MS was then performed to identify protein spots that were differentially expressed between the two groups and western blot analysis was used to validate the expressional change of protein demonstrated by proteomics. Our proteomics and bioinformatics analysis identified 11 proteins differentially expressed between DR and control groups. These proteins are linked to biological networks associated with nutrition transport, microstructure reorganization, angiogenesis, anti-oxidation, and neuroprotection. The data may provide potential AH biomarkers and susceptibility factors for predicting DR development, and provide an insight into the underlying pathophysiological mechanisms of DR. This article is part of a Special Issue entitled: Proteomics: The clinical link.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3fb2a8b2f19e27cf4d443ac32a39ff2cTest
https://doi.org/10.1016/j.jprot.2011.12.006Test

المؤلفون: Ching-Wu Hsia, Han-Min Chen, Chih-Yuan Wang, Tien-Chun Chang, Hao-Ai Shui

المصدر: Journal of Proteomics. 75:1170-1180

مصطلحات موضوعية: Proteomics, Protein Folding, Blotting, Western, FLOT1, Biophysics, DNA Fragmentation, Biology, Protein degradation, Biochemistry, Downregulation and upregulation, medicine, Humans, Lovastatin, Thyroid Neoplasms, Cloning, Molecular, Anaplastic thyroid cancer, Thyroid cancer, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Computational Biology, Membrane Proteins, medicine.disease, Molecular biology, Cell biology, Gene Expression Regulation, Neoplastic, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Cancer cell, Hydroxymethylglutaryl-CoA Reductase Inhibitors, Signal transduction, Signal Transduction

الوصف: Lovastatin (lova), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, can induce differentiation in cancer cells at low concentration, thus having potential to be used as an auxiliary agent in cancer therapy. However, biological networks associated with the differentiation effect of lova have not been elucidated. To investigate molecular mechanisms of lova, the present study was aimed at proteomics and bioinformatics analyses on anaplastic thyroid cancer cell line ARO differentiated with low concentration of lova. Thyroid differentiation was induced by treating ARO cells with 25 μM of lova and confirmed by checking upregulation of some thyroid differentiation markers. Gel-based proteomics analysis was then performed to identify proteins differentially expressed between undifferentiated and lova-differentiated ARO cells. Bioinformatics analysis was finally performed to estimate biological networks regulated by lova. Our results showed that lova impacted on proteins involved in protein folding, biomolecule metabolism, signal transduction, protein expression and protein degradation. Specifically, transfecting ARO cells with plasmid DNA encoding flotillin 1 (FLOT1) up-regulated the thyroid differentiation markers, indicating that FLOT1 might at least partially mediate the lova-induced thyroid differentiation. These data may shed light on the mechanism underlying lova-induced re-differentiation of thyroid cancer, and give a rationale for clinical use of lova as an auxiliary agent in cancer therapy.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7b8f291b405b57ded426079f91811e41Test
https://doi.org/10.1016/j.jprot.2011.10.029Test

المؤلفون: Yen Ru Lin, Han Min Chen, Hui Kang Liu, Wen Lin Lai, Nai-Kuei Huang, Yi Chao Lee, Chuen Lin Huang, Wei Chen Liao

المصدر: Biochemical and Biophysical Research Communications. 354:96-101

مصطلحات موضوعية: Programmed cell death, Thioredoxin reductase, Biophysics, Down-Regulation, Apoptosis, Biology, medicine.disease_cause, PC12 Cells, Biochemistry, Methamphetamine, chemistry.chemical_compound, Thioredoxins, medicine, Animals, Molecular Biology, Dose-Response Relationship, Drug, Peroxiredoxins, Cell Biology, Meth, respiratory system, Molecular biology, Rats, Cell biology, Gene Expression Regulation, Neoplastic, Peroxidases, chemistry, Thioredoxin, Peroxiredoxin, Oxidative stress, Signal Transduction, medicine.drug

الوصف: Methamphetamine (METH) is an abusive psychostimulant that induces neuronal cell death/degeneration in experimental animals and humans. METH-induced apoptosis in rat pheochromocytoma cells was utilized to study the neurotoxic mechanism. During METH intoxication, we found that peroxiredoxins and thioredoxins/thioredoxin reductases (peroxiredoxin reducing systems) which are known to prevent oxidative stress and apoptosis were differentially downregulated and upregulated, respectively. We also found not only the free radicals but also the oxidative forms of peroxiredoxin and thioredoxin were increased, indicating the dysfunction of these enzymes. Thus, METH-induced differential regulation and oxidation of peroxiredoxins and thioredoxin may be an important mechanism for apoptosis.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::17371c3282b494ff2fbeab4c67dbb00eTest
https://doi.org/10.1016/j.bbrc.2006.12.138Test

المؤلفون: Jing-Yi Nong, Po-Ku Chen, Yi-Fang Cheng, Cheng-Yu Chao, Han-Min Chen, Jiun-Tsai Lin, Chun-Fang Huang, Guang-Huar Young

المصدر: Journal of proteomics. 120

مصطلحات موضوعية: biology, Proteome, Activator (genetics), Glucose uptake, Adenine, Biophysics, Glucose transporter, AMPK, AMP-Activated Protein Kinases, Biochemistry, Gene Expression Regulation, Enzymologic, Enzyme Activation, Insulin receptor, Mice, Glucose, Downregulation and upregulation, biology.protein, NIH 3T3 Cells, Animals, Glycolysis, Protein kinase A

الوصف: AMP-activated protein kinase (AMPK) is a metabolic master switch maintaining the energy homeostasis in cells and thought to modulate cellular response to stresses. Adenine as well as a pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), induced the phosphorylation of AMPK and acetyl-CoA carboxylase in NIH/3T3 cells. Administration of adenine or AICAR increased the expression and translocation of glucose transporter 4, enhanced the cellular glucose uptake, and elevated the intracellular ATP level. To better understand the proteomic changes in response to exogenous adenine treatment, we performed two-dimensional difference gel electrophoresis (2DE-DIGE) and grouped protein spots with similar intensities prior to MS analysis. These process allowed us to exclude these constant expressed proteins, reduce the coverage from abundant signals and increase the identification of middle/lower expressed proteins. Bioinformatics analysis on the proteomic alterations suggested that both of adenine and AICAR could induce up-regulation of a panel of proteins associated with glucose metabolism. We also found that adenine upregulated expression of the glycolytic enzyme, hexokinase 2, indicating a link between adenine and AMPK-mediated glycolysis. Taken together, by demonstrating the adenine-mediated proteome changes in NIH/3T3 cells, our study provides useful information for the characteristics of adenine-induced AMPK activation and development of efficient AMPK activator. Biological significance AMPK is a fuel sensing enzyme that responds to a central role of energy homeostasis and contributes to the acceleration of insulin signaling. Recently, we have shown that exogenous adenine exerted anti-inflammatory effects through activation of AMPK, suggesting the treatment is a potent therapeutic strategy against hyperglycemia. Adenine had similar effects with 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) in modulating glucose uptake via AMPK-mediated signaling. In this study, we performed a 2DE-DIGE/MS-based approach to investigate the mechanism of exogenous adenine in NIH/3T3 cells. Our results provide evidence of a novel role for adenine in AMPK-mediated signaling and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::d152508f18fbf3b0be0fa5a2bec291b8Test
https://pubmed.ncbi.nlm.nih.gov/25797921Test

المؤلفون: Hao-Ai Shui, Pei-Hsiu Chen, Han-Min Chen, Min-Jen Tseng, Ann Chen, Hung-Te Liu, Yung-Fu Wu, Chih-Yuan Wang, Ming-Yi Ho, Ching-Wu Hsia, Tzu-Hao Huang

المصدر: Journal of proteomics. 74(12)

مصطلحات موضوعية: Male, Proteomics, endocrine system diseases, Paclitaxel, Systems biology, Biophysics, Apoptosis, macromolecular substances, Biology, Biochemistry, Rats, Sprague-Dawley, Organ Culture Techniques, medicine, Animals, Dose-Response Relationship, Drug, organic chemicals, food and beverages, Computational Biology, Alopecia, Metabolism, Dermis, medicine.disease, Antineoplastic Agents, Phytogenic, Pathophysiology, Cell biology, Rats, Hair loss, Vibrissae, Proteome, Protein folding, Calcium

الوصف: Dermal papilla (DP) cells play a regulatory role in hair growth, and also play a role in alopecia (hair loss). However, effects of taxol, which is a widely used chemotherapy drug, on DP cells remain unclear, despite that theoretically taxol can impact on DP cells to contribute to taxol-induced alopecia. To better understand pathophysiology of taxol-induced damage in DP cells, morphological and biochemical analyses were performed to check whether taxol can cause apoptosis in cultured DP cells or not. If it can, proteomics and bioinformatics analyses were then performed to investigate the protein networks which are impacted by the taxol treatment. Our data showed that taxol can cause apoptotic damage in DP cells in a concentration-dependant manner, as demonstrated by various apoptotic markers. Proteomic analysis on DP cells treated with the lowest apoptosis-inducible concentration of taxol revealed that taxol can affect expression of proteins involved in Ca2+-regulated biological processes, vesicles transport, protein folding, reductive detoxification, and biomolecules metabolism. Furthermore, bioinformatics analysis indicated that taxol can impact on multiple biological networks. Taken together, this biochemical, proteomics, and bioinformatics data may give an insight into pathophysiology of taxol-induced damage in DP cells and shed light on mechanisms underlying taxol-induced alopecia.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ac7d84770996664efc55dedbabf3783dTest
https://pubmed.ncbi.nlm.nih.gov/21989266Test

المؤلفون: Chien-Chang Yen, Wen-Huei Tsui, Hui-Chung Wu, Han-Min Chen

المصدر: Analytical biochemistry. 396(1)

مصطلحات موضوعية: Commercial software, Standard test image, Computer science, business.industry, Binary image, Biophysics, Analytical chemistry, Proteins, Image processing, Signal Processing, Computer-Assisted, Cell Biology, Biochemistry, Grayscale, Visualization, Transformation (function), Image Processing, Computer-Assisted, Animals, Computer vision, Electrophoresis, Polyacrylamide Gel, Artificial intelligence, Image analysis, business, Molecular Biology, Algorithms, Software

الوصف: Currently, results of gel electrophoresis are commonly documented in digital formats by image acquisition instruments. In this study, gel images tuned by a common image processing software package, Photoshop, were assessed to understand the transforming algorithms and their impacts on quantitative analysis. TotalLab 100, an electrophoresis gel image analysis software package, was applied for image quantitation and evaluation. The three most frequently used image tuning functions—adjustments of the brightness, contrast, and grayscale span (level) of images—were investigated using both data generated from a standard grayscale tablet and an actual electrophoresis gel image. The influences of these procedures were analyzed for the grayscale transformation between the input and output images. Although all three procedures differentially improved the visualization of the input image, adjusting the contrast of images disrupted the quantitative information because of its nonlinear transforming algorithm. Under certain conditions, adjusting the brightness or the level of images could preserve the quantitative information because of the linear transforming algorithms. It was found that when the minimum and maximum grayscales of a gel image were recognized, using a commercial software package to maximally stretch the level may significantly improve the quality of a gel image without jeopardizing quantitative analysis.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8e14279af92c53fbd14b81f39404ae03Test
https://pubmed.ncbi.nlm.nih.gov/19733146Test

المؤلفون: Sheng-He Huang, Ambrose Jong, Han-Min Chen, Chu-Hua Wu, Luo Feng, Shibo Jiang

المصدر: Biochemical and biophysical research communications. 356(4)

مصطلحات موضوعية: Membrane ruffling, Biophysics, Blood–brain barrier, Gp41, Biochemistry, law.invention, Microbiology, Structure-Activity Relationship, law, medicine, Cell Adhesion, Humans, Molecular Biology, Pathogen, Actin, Cells, Cultured, Cryptococcus neoformans, biology, Microcirculation, Brain, Endothelial Cells, Cell Biology, biology.organism_classification, HIV Envelope Protein gp41, Protein Structure, Tertiary, medicine.anatomical_structure, Ectodomain, Cerebrovascular Circulation, Recombinant DNA

الوصف: Cryptococcus neoformans infection has significantly increased recently, particularly in AIDS patients and immunocompromised individuals. C. neoformans has a predilection to the brain, resulting in devastating meningoencephalitis. We have previously shown the invasion of C. neoformans into the human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. Here, we demonstrated that C. neoformans invasion of HBMEC was enhanced by HIV-1 gp41 protein. Peptide mapping defined its functional domain around the disulfide-bond linkage of gp41 molecule (a.a. 579-611). Recombinant protein gp41-I90 (a.a. 550-639) can also enhance the binding activity. The enhancement of C. neoformans binding to HBMEC is a strain-independent manner, suggesting that gp41 ectodomain peptide exerts its function directly on HBMEC. Importantly, the enhancement could be observed in mouse animal model. Our results suggest that HIV-1 gp41 ectodomain and C. neoformans may follow a similar invasion mechanism, possibly actin reorganization and/or membrane activation, during pathogen infections on HBMEC.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e2f3b9c577dd814535d24c422a72dd88Test
https://pubmed.ncbi.nlm.nih.gov/17400192Test