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Journal articles on the topic '2D electrophoresis display'
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1
Spandidos, Athanasia, and Terence H. Rabbitts. "Sub-proteome Differential Display: Single Gel Comparison by 2D Electrophoresis and Mass Spectrometry." Journal of Molecular Biology 318, no. 1 (April 2002): 21–31. http://dx.doi.org/10.1016/s0022-2836(02)00052-9.
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Michieletto, Davide, Davide Marenduzzo, and Enzo Orlandini. "Topological patterns in two-dimensional gel electrophoresis of DNA knots." Proceedings of the National Academy of Sciences 112, no. 40 (September 8, 2015): E5471—E5477. http://dx.doi.org/10.1073/pnas.1506907112.
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Gel electrophoresis is a powerful experimental method to probe the topology of DNA and other biopolymers. Although there is a large body of experimental work that allows us to accurately separate different topoisomers of a molecule, a full theoretical understanding of these experiments has not yet been achieved. Here we show that the mobility of DNA knots depends crucially and subtly on the physical properties of the gel and, in particular, on the presence of dangling ends. The topological interactions between these and DNA molecules can be described in terms of an “entanglement number” and yield a nonmonotonic mobility at moderate fields. Consequently, in 2D electrophoresis, gel bands display a characteristic arc pattern; this turns into a straight line when the density of dangling ends vanishes. We also provide a novel framework to accurately predict the shape of such arcs as a function of molecule length and topological complexity, which may be used to inform future experiments.
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Rezaei, Ali, Saeme Asgari, Samira Komijani, Seyedeh Narjes Sadat, Jean-Marc Sabatier, Davood Nasrabadi, Kamran Pooshang Bagheri, et al. "Discovery of Leptulipin, a New Anticancer Protein from theIranian Scorpion, Hemiscorpius lepturus." Molecules 27, no. 7 (March 22, 2022): 2056. http://dx.doi.org/10.3390/molecules27072056.
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Cancer is one of the leading causes of mortality in the world. Unfortunately, the present anticancer chemotherapeutics display high cytotoxicity. Accordingly, the discovery of new anticancer agents with lower side effects is highly necessitated. This study aimed to discover an anticancer compound from Hemiscorpius lepturus scorpion venom. Bioactivity-guided chromatography was performed to isolate an active compound against colon and breast cancer cell lines. 2D electrophoresis and MALDI-TOF were performed to identify the molecule. A partial protein sequence was obtained by mass spectrometry, while the full-length was deciphered using a cDNA library of the venom gland by bioinformatics analyses and was designated as leptulipin. The gene was cloned in pET-26b, expressed, and purified. The anticancer effect and mechanism action of leptulipin were evaluated by MTT, apoptosis, and cell cycle assays, as well as by gene expression analysis of apoptosis-related genes. The treated cells displayed inhibition of cell proliferation, altered morphology, DNA fragmentation, and cell cycle arrest. Furthermore, the treated cells showed a decrease in BCL-2 expression and an increase in Bax and Caspase 9 genes. In this study, we discovered a new anticancer protein from H. lepturus scorpion venom. Leptulipin showed significant anticancer activity against breast and colon cancer cell lines.
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Carra, G., and R. S. Accolla. "Structural analysis of human Ia antigens reveals the existence of a fourth molecular subset distinct from DP, DQ, and DR molecules." Journal of Experimental Medicine 165, no. 1 (January 1, 1987): 47–63. http://dx.doi.org/10.1084/jem.165.1.47.
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Structural analysis by two-dimensional peptide maps (2D-PM) of the human Ia molecular pool expressed on the cell surface of two distinct lymphoblastoid cell line, LG-2 and Raji, revealed the existence of a novel MHC class II molecular heterodimer that differs at the level of both alpha and beta subunits from the previously described DP, DQ, and DR antigens. These differences were also seen at the level of two-dimensional electrophoresis (2D-PAGE) of biosynthetically labeled intact molecules, although to a lesser extent, due to the intrinsic limitations of this technique in resolving fine structural differences. We have designated this new class II antigen as the fourth Ia subset. The fourth Ia subset seems to represent a small proportion of the human Ia pool. Comparative analysis by 2D-PM of the two cell lines showed the presence of structural variations in the alpha chains of the fourth Ia subset, suggesting the existence of polymorphism for these subunits. Cell surface iodination did not show appreciable labeling of the fourth subset beta chain in LG-2 cells, and this prevented analysis of the structural polymorphism of this subunit. Furthermore, for the first time, we have shown that DP alpha chains display distinct peptide maps in LG-2 and Raji cells, thus suggesting the presence of structural polymorphism for these Ia subunits also. The DQ1 alpha and beta allelic products present in LG-2 cells (DQ homozygous) did not show appreciable structural variation when compared with the homologous allelic products present in Raji cells (DQ heterozygous). Finally, we have confirmed the absence of polymorphism for the DR alpha subunits. By 2D-PM, relatively low structural variation was instead found for the highly polymorphic DR beta subunits expressed in the two cell lines, suggesting that cell surface iodination preferentially labels constant domains of DR beta chains.
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Müller, Virginia, Gustavo Bonacci, Carlos Batthyany, María V. Amé, Fernando Carrari, Jorge Gieco, and Ramón Asis. "Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors." Phytopathology® 107, no. 4 (April 2017): 474–82. http://dx.doi.org/10.1094/phyto-09-16-0346-r.
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Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.
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Li, Shenjie, Wei Xiang, Junjie Tian, Haorun Wang, Shuiwang Hu, Ke Wang, Ligang Chen, Changren Huang, and Jie Zhou. "Bone Marrow-Derived Mesenchymal Stem Cells Differentially Affect Glioblastoma Cell Proliferation, Migration, and Invasion: A 2D-DIGE Proteomic Analysis." BioMed Research International 2021 (February 11, 2021): 1–13. http://dx.doi.org/10.1155/2021/4952876.
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Bone marrow-derived mesenchymal stem cells (BM-MSCs) display high tumor tropism and cause indirect effects through the cytokines they secrete. However, the effects of BM-MSCs on the biological behaviors of glioblastoma multiforme remain unclear. In this study, the conditioned medium from BM-MSCs significantly inhibited the proliferation of C6 cells ( P < 0.05 ) but promoted their migration and invasion ( P < 0.05 ). Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) proteomic analysis revealed 17 proteins differentially expressed in C6 cells exposed to the BM-MSC-conditioned medium including five upregulated proteins and 12 downregulated proteins. Among these, six differentially expressed proteins (Calr, Set, Oat, Npm1, Ddah1, and Tardbp) were closely related to cell proliferation and differentiation, and nine proteins (Pdia6, Sphk1, Anxa4, Vim, Tuba1c, Actr1b, Actn4, Rap2c, and Tpm2) were associated with motility and the cytoskeleton, which may modulate the invasion and migration of tumor cells. Above all, by identifying the differentially expressed proteins using proteomics and bioinformatics analysis, BM-MSCs could be genetically modified to specifically express tumor-suppressive factors when BM-MSCs are to be used as tumor-selective targeting carriers in the future.
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Raffenne, Jérôme, Fernando A. Martin, Rémy Nicolle, Marina Konta, Yuna Blum, Jérôme Torrisani, Francesco Puleo, et al. "Pancreatic Ductal Adenocarcinoma Arising in Young and Old Patients Displays Similar Molecular Features." Cancers 13, no. 6 (March 11, 2021): 1234. http://dx.doi.org/10.3390/cancers13061234.
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Pancreatic ducal adenocarcinoma is classically diagnosed in the 7th decade, but approximately 10% of patients are diagnosed under 55 years (y.o.). While the genomic and transcriptomic landscapes of late-onset tumors (LOT) have been described, little is known about early-onset tumors (EOT). Ageing is known to impact DNA methylation and proteome integrity through carbonylation-related oxidative damages. We therefore aimed to assess the global molecular features of EOT. We compared 176 EOT (≤55 y.o.) and 316 LOT (≥70 y.o.) from three distinct surgical cohorts at the clinical/genomic/epigenomic/transcriptomic level. Furthermore, we assessed oxidative stress responses and oxidative proteome damages using 2D gel electrophoresis followed by mass spectrometry protein identification. There was no consistent clinical difference between EOT and LOT across the three cohorts. The mutational landscape of key driver genes and the global methylation profile were similar in the two groups. LOT did display age-related features such as enriched DNA repair gene signatures and upregulation of oxidative stress defenses together with increased proteome carbonylation. However, these age-related differences were more preeminent in non-tumor tissues while tumor proteome and proteome damages were fairly comparable. In conclusion, this multi-omics comparison showed that EOT harbor a comparable molecular profile to that of LOT.
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Burkova, Evgeniya E., Alina E. Grigor’eva, Dmitrii V. Bulgakov, Pavel S. Dmitrenok, Valentin V. Vlassov, Elena I. Ryabchikova, Sergey E. Sedykh, and Georgy A. Nevinsky. "Extra Purified Exosomes from Human Placenta Contain an Unpredictable Small Number of Different Major Proteins." International Journal of Molecular Sciences 20, no. 10 (May 16, 2019): 2434. http://dx.doi.org/10.3390/ijms20102434.
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Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30–100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins’ tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11–13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.
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Ni, Tie-Hua, William F. McDonald, Irene Zolotukhin, Thomas Melendy, Shou Waga, Bruce Stillman, and Nicholas Muzyczka. "Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection." Journal of Virology 72, no. 4 (April 1, 1998): 2777–87. http://dx.doi.org/10.1128/jvi.72.4.2777-2787.1998.
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ABSTRACT We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and ɛ, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase ɛ for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.
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Smith, Marjorie A., Satbinder K. Bains, Joanna C. Betts, Ernest H. S. Choy, and Edward D. Zanders. "Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4." Clinical Diagnostic Laboratory Immunology 8, no. 1 (January 1, 2001): 105–11. http://dx.doi.org/10.1128/cdli.8.1.105-111.2001.
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ABSTRACT Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern of plasma-derived glycoproteins. The lowest level of detection was approximately 0.2 ng from a total of 50 μg of protein loaded. Most of the proteins could be identified on the basis of pI and molecular weight when compared with plasma protein maps on the World Wide Web. Unknown proteins were characterized by mass spectrometry of tryptic digests and by comparison with peptide databases. Synovial fluids from patients with rheumatoid arthritis were analyzed using this technique. Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent removal of synovial fluid and additional administration of antibody were carried out at different times thereafter. Changes in levels of acute-phase proteins were quantified by densitometry of silver-stained 2D polyacrylamide gels. Other parameters of disease progression such as serum C-reactive protein and physician's global assessment of clinical condition were used for comparison. In this way, changes in acute-phase proteins towards normal levels, as measured by 2D polyacrylamide gel electrophoresis, could be correlated with clinical improvement and conventional clinical chemistry measurements. Thus, the system can be used for quantitative analysis of protein expression in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.
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Shrivastava, Rahul, Bhakti Basu, Ashwini Godbole, M. K. Mathew, Shree K. Apte, and Prashant S. Phale. "Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86." Microbiology 157, no. 5 (May 1, 2011): 1531–40. http://dx.doi.org/10.1099/mic.0.047191-0.
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Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [14C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain’s ability to utilize aromatics and organic acids over glucose.
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Pötgens, Andy J. G., Ulrike Schmitz, Peter Kaufmann, and Hans-Georg Frank. "Monoclonal Antibody CD133–2 (AC141) Against Hematopoietic Stem Cell Antigen CD133 Shows Crossreactivity with Cytokeratin 18." Journal of Histochemistry & Cytochemistry 50, no. 8 (August 2002): 1131–34. http://dx.doi.org/10.1177/002215540205000814.
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CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133–2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133–2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133–2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133–2/AC141 in IHC.
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Komatsu, Setsuko, Myeong W. Oh, Hee Y. Jang, Soo J. Kwon, Hye R. Kim, Jung H. Ko, Sun H. Woo, and Yohei Nanjo. "Proteomic Analyses of Soybean Root Tips During Germination." Protein & Peptide Letters 21, no. 12 (November 5, 2014): 1308–19. http://dx.doi.org/10.2174/0929866521666140526152426.
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Plant root systems form complex networks with the surrounding soil environment and are controlled by both internal and external factors. To better understand the function of root tips of soybean during germination, three proteomic techniques were used to analyze the protein profiles of root tip cells. Proteins were extracted from the root tips of 4-dayold soybean seedlings and analyzed using two-dimensional (2D) gel electrophoresis-based proteomics, SDS-gel based proteomics, and gel-free proteomics techniques. A total of 121, 862, and 341 proteins were identified in root tips using the 2D gel-based, SDS gel-based, and gel-free proteomic techniques, respectively. The proteins identified by 2D gel-based proteomic analysis were predominantly localized in the cytoplasm, whereas nuclear-localized proteins were most commonly identified by the SDS gel-based and gel-free proteomics techniques. Of the 862 proteins identified in the SDS gelbased proteomic analysis, 190 were protein synthesis-related proteins. Furthermore, 24 proteins identified using the 2Dgel based proteomic technique shifted between acidic and basic isoelectric points, and 2 proteins, heat shock protein 70.2 and AAA-type ATPase, displayed two different molecular weights at the same isoelectric point. Taken together, these results suggest that a number of proteins related to protein synthesis and modification are activated in the root tips of soybean seedlings during germination.
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Konidaris, Konstantis F., Rigini Papi, Eugenia Katsoulakou, Catherine P. Raptopoulou, Dimitrios A. Kyriakidis, and Evy Manessi-Zoupa. "Synthesis, Crystal Structures, and DNA Binding Properties of Zinc(II) Complexes with 3-Pyridine Aldoxime." Bioinorganic Chemistry and Applications 2010 (2010): 1–7. http://dx.doi.org/10.1155/2010/803424.
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The employment of 3-pyridine aldoxime, (3-py)CHNOH, in ZnIIchemistry has afforded two novel compounds: [Zn(acac)2{(3-py)CHNOH}]⋅H2O(1⋅H2O) [where acac-is the pentane-2,4-dionato(-1) ion] and [Zn2(O2CMe)4{(3-py)CHNOH}2] (2). Complex1⋅H2Ocrystallizes in the monoclinic space groupP21/n. The ZnIIion is five-coordinated, surrounded by four oxygen atoms of two acac-moieties and by the pyridyl nitrogen atom of the (3-py)CHNOH ligand. Molecules of1interact with the water lattice molecules forming a 2D hydrogen-bonding network. Complex2crystallizes in the triclinicP-1space group and displays a dinuclear paddle-wheel structure. Each ZnIIexhibits a perfect square pyramidal geometry, with four carboxylate oxygen atoms at the basal plane and the pyridyl nitrogen of one monodentate (3-py)CHNOH ligand at the apex. DNA mobility shift assays were performed for the determination of thein vitroeffect of both complexes on the integrity and the electrophoretic mobility of pDNA.
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De Oliveira Martins, Carlo, Keity Souza Santos, Frederico Moraes Ferreira, Priscila Camillo Teixeira, Pablo Maria Alberto Pomerantzeff, Carlos M. A. Brandão, Roney Orismar Sampaio, et al. "Distinct Mitral Valve Proteomic Profiles in Rheumatic Heart Disease and Myxomatous Degeneration." Clinical Medicine Insights: Cardiology 8 (January 2014): CMC.S17622. http://dx.doi.org/10.4137/cmc.s17622.
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Rheumatic heart disease (RHD) affects heart-valve tissue and is the most serious consequence of group A Streptococcus infection. Myxomatous degeneration (MXD) is the most frequent valvopathy in the western world. In the present work, key protein expression alterations in the heart-valve tissue of RHD and MXD patients were identified and characterized, with controls from cadaveric organ donors. Proteins were separated by two-dimensional (2D)-electrophoresis and identified by mass spectrometry. We found 17 differentially expressed protein spots, as compared to control samples. We observed an increased expression of ASAP-2 in the RHD patients’ valves, while collagen-VI, haptoglobin-related protein, prolargin, and cartilage oligomeric protein showed reduced expression. Valve tissue of MXD patients, on the other hand, presented lower expression of annexin-Al and A2, septin-2, SOD (Cu/Zn), and transgelin. Tissue samples from both valvopathies displayed higher expression of apolipoprotein-Al. Biglycan was downexpressed in both diseases. Vimentin and lumican showed higher expression in RHD and lower in MXD. These results suggest that key pathogenetic mechanisms are intrinsically distinct in RHD and MXD.
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Raina (PhD), Varshiesh, and Konika Razdan (PhD). "A XENOGENIC IMMUNE RESPONSE TOWARDS STZ-RINM5F CELLS REVEALS CYTOKERATIN18 AS A NOVEL IMMUNOGENIC ANTIGEN THAT MAY PREDISPOSE TOWARDS TYPE-1 DIABETES." International Journal of Advanced Research 10, no. 07 (July 31, 2022): 651–69. http://dx.doi.org/10.21474/ijar01/15083.
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Studies suggest certain stimuli like apoptosis behind the aberrant expression of circulating specific auto-antibodies in Type-1 diabetic patients. In the present study, we investigated whether Streptozotocin (a diabetogenic compound) induced apoptosis in Rat RINm5f Insulinoma cells can expose immunogenic cryptic antigens. A time course treatment of RINm5f cells with 4 mM Streptozotocin revealed 6hrs as minimal time period to induce apoptosis without much effect on viability.Subsequent immunization of mice with the untreated and Streptozotocin treated RINm5f cells revealed highly reactive sera from 6hr STZ treated cells. Hybridoma technique showed a highly reactive clone named sup160which secretedIgG1 type monoclonal antibody.Parallely, we show that 6hr STZ-RINm5f challenged mice displayed both humoral and cellular immune response, as shown by increased presence of IgG1 / IgG2a subclass of antibodies and overexpression of IFNγ, IL-4 and TNFα in splenocytes. FACS and confocal imaging further established the reactivity of sup160 antibody with RINm5f cell surface antigen. The antigen was identified to be cytokeratin18 protein as detected by 2D gel electrophoresis and mass spectrometry. In conclusion, we have successfully established a novel xenogenic mice model system for identification of new auto-antigen in Type-1 diabetes, although further studies are warranted in human subjects.
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Lucas, Maria, Eugenia Mato, Silvia Barceló-Batllori, Ramon Gomis, and Anna Novials. "Proteomics Characterization of the Secretome from Rat Pancreatic Stellate Cells with ATP-Binding Cassette Transporters (ABCG2) and NCAM Phenotype." ISRN Cell Biology 2013 (October 31, 2013): 1–18. http://dx.doi.org/10.1155/2013/828060.
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We have previously reported the identification of a pancreata mitoxantrone-resistant cell population which expressed the ABCG2 transporter with a pancreatic stellate cells phenotype (PaSC) and ability of secreting insulin after inducing their differentiation. The characterization of the secretome of this cell population by two-dimensional electrophoresis (2D) coupled with mass spectrometry MALDI-TOF was able to identify seventy-six protein spots involved in different cellular processes: development/differentiation, proteases, immune response, and other. Moreover, Ingenuity Pathway Analysis displayed several significant networks and TGFβ1 molecule was identified as a central node of one of them. The effect of this active molecule secreted in the conditioned medium was investigated in ductal cell line (ARIP). The results showed that the conditioned medium inhibited their proliferation without affecting their cell viability. Additionally, they showed an upregulation of PDX1 and downregulation of CK19. The rate of ARIP cell proliferation was recovered, but no effects on the gene expression were observed after using TGFβ1-neutralising antibody. Proteins associated with cell growth, development and differentiation such as PEDF, LIF, and Wnt5b, identified in the secretome, could be involved in the observed transcription changes. These finding may suggest a new paracrine action of PaSCs involved in the proliferation and differentiation pathways not yet identified.
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Viale, Mariana Noelia, Gabriela Echeverria-Valencia, Pablo Romasanta, María Laura Mon, Marisa Fernandez, Emilio Malchiodi, María Isabel Romano, Andrea Karina Gioffré, and María de la Paz Santangelo. "Description of a Novel Adhesin ofMycobacterium aviumSubsp.paratuberculosis." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/729618.
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The binding and ingestion ofMycobacterium aviumsubsp.paratuberculosis(MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within theμM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.
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Wang, Yan, Jiaxin Liu, and Fancheng Lin. "A Photoelectrochemical Sensor for the Sensitive Detection of Cysteine Based on Cadmium Sulfide/Tungsten Disulfide Nanocomposites." Nanomaterials 14, no. 5 (February 27, 2024): 427. http://dx.doi.org/10.3390/nano14050427.
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In this work, a CdS-nanoparticle-decorated WS2 nanosheet heterojunction was successfully prepared and first used to modify ITO electrodes for the construction of a novel photoelectrochemical sensor (CdS/WS2/ITO). The thin-film electrode was fabricated by combining electrophoretic deposition with successive ion layer adsorption and reaction techniques. The results indicated that the synthesized heterojunction nanomaterials displayed excellent photoelectrochemical performance which was much better than that of pristine CdS nanoparticles and 2D WS2 nanosheets. Owing to the formation of the surface heterojunction and the effective interfacial electric field, the enhanced separation of photogenerated electron–hole pairs led to a remarkable improvement in the photoelectrochemical activity of CdS/WS2/ITO. This heterojunction architecture can protect CdS against photocorrosion, resulting in a stable photocurrent. Based on the specific recognition between cysteine and CdS/WS2/ITO, through the specificity of Cd-S bonds, a visible-light-driven photoelectrochemical sensor was fabricated for cysteine detection. The novel photoelectrochemical biosensor exhibited outstanding analytical capabilities in detecting cysteine, with an extremely low detection limit of 5.29 nM and excellent selectivity. Hence, CdS-WS2 heterostructure nanocomposites are promising candidates as novel advanced photosensitive materials in the field of photoelectrochemical biosensing.
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Feng, Bo, Francesco Sestili, Stefania Masci, Benedetta Margiotta, Zhibin Xu, Zujun Yang, Chao Xiang, Chunhong Zhou, Domenico Lafiandra, and Tao Wang. "The Chinese bread wheat cultivar Xiaoyanmai 7 harbours genes encoding a pair of novel high-molecular-weight glutenin subunits inherited from cereal rye." Crop and Pasture Science 67, no. 1 (2016): 29. http://dx.doi.org/10.1071/cp15191.
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The high-molecular-weight glutenin subunits (HMW-GS) represent a major component of the endosperm storage protein in the grains of wheat and its related species. Their technological importance results from their ready formation of intermolecular disulfide bonds, which underlie much of the visco-elasticity displayed by gluten and hence the processing quality of the flour. Here, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the Chinese wheat cultivar Xiaoyanmai 7 formed four distinct HMW-GS, two of which are likely the product of a known allele at the Glu-B1 locus, whereas the other two did not match any known HMW-GS. A combined analysis based on reversed-phase high-performance liquid chromatography (RP-HPLC), N-terminal sequencing and mass spectrometry confirmed that the two novel proteins were genuine HMW-GS. Inspection of the DNA sequences showed that one of the novel HMW-GS was encoded by an x-type and the other by a y-type secalin gene. A karyotypic analysis confirmed that six of the seven pairs of Xiaoyanmai 7’s D genome chromosomes (the exception was chromosome 2D) had been replaced by rye chromosomes. The y-type HMW secalin present in Xiaoyanmai 7 differed from the standard By and Dy HWM-GS by the presence of an additional cysteine residue in its C-terminal domain.
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Wang, Ruomei, Jisu Wu, Xiong Deng, Dongmiao Liu, and Yueming Yan. "Drought-responsive protein identification in developing grains of a wheat–Haynaldia villosa 6VS/6AL translocation line." Crop and Pasture Science 69, no. 12 (2018): 1182. http://dx.doi.org/10.1071/cp18303.
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Drought is a widespread abiotic stress that has a detrimental effect on both yield and quality of wheat. Discovery and utilisation of drought-resistant gene resources from wheat-related species may help to mitigate effects of drought and decrease yield loss. In this study, we used a comparative proteome approach to identify potential drought-resistance proteins from a wheat (Triticum aestivum L.)–Haynaldia villosa (L.) Schur 6VS/6AL translocation line. Drought experiments showed that introgression of the H. villosa 6VS chromosome short arm into common wheat cultivar Yangmai 5 through 6VS/6AL translocation led to better drought resistance. Two-dimensional difference gel electrophoresis (2D-DIGE) identified 99 differentially accumulated protein (DAP) spots in the wheat–H. villosa 6VS/6AL translocation line, 42 of which were specifically present or showed a significantly upregulated accumulation. Of these, 20 DAPs representing 19 unique proteins in the wheat–H. villosa 6VS/6AL translocation line were upregulated under drought stress. These proteins were mainly involved in defence–stress, energy metabolism, carbon metabolism, nitrogen metabolism, and protein metabolism or folding. Protein–protein interaction analysis of key DAPs displayed a complex interaction network that synergistically regulated drought response. Dynamic transcriptional expression analysis revealed the differential expression of six key DAP genes involved in drought-stress response in the protein–protein interaction network. Our results indicated that H. villosa may have gene resources for wheat drought-resistance improvement.
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Yang, Mengdi, Jing Sun, Zhiyu Wang, Rui Zhang, YIfeng Gu, and Hui Zhao. "Differential expression proteins in lung adenocarcinoma with bone metastasis: Correlation of ALDH2 and ENO1 with prognosis." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e20534-e20534. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20534.
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e20534 Background: The prognosis of lung adenocarcinoma (LUAD) with bone metastases (BM) is still poor. It is increasingly demanded to find key proteins to solve the problem. Methods: First, we collected three groups of bone tissues: normal, osteosarcoma, and LUAD with BM from Shanghai Sixth People's Hospital. Each group had five patients. Two-dimensional gel electrophoresis (2D-E) was used to find proteins with significant differences, then Maldi-tof-tof mass spectrometry was used to identify the proteins. Second, immunohistochemical (IHC) was used to check the expression of proteins in 28 BM patients and 9 LUAD patients. Real-Time PCR (QPCR), western boltting (WB) were further used to check the expression of para-LUAD, LUAD and BM. Last, we downloaded RNA-seq and clinical datas from the Cancer Genome Atlas(TCGA) database to further verify differentially expressed genes using ‘DESeq’ package and explored their relationship with survival using ‘survival’ package in R. Kaplan-Meier survival plot and the hazard ratio and logrankp was calculated (www.kmplot.com/lung). Results: We got 26 matched proteins from 2D-E, then selected Aldehyde Dehydrogenase 2 Family (Mitochondrial) (ALDH2) and Enolase-1 (ENO1). In IHC analysis, strong-stained ALDH2 showed OR = 0.568 (95 % confidence interval [CI], 0.317-1.019), p= 0.036, strong-stained ENO1 OR = 1.929 (95% CI, 0.915-4.066), p= 0.023, were associated with bone metastastic incidences in LUAD with statistically significantly difference. QPCR, WB validated that ALDH2 had lowest expression in BM, followed by LUAD samples, and para-LUAD highest. However, ENO1 displayed the opposite tendency . In TCGA LUAD RNA-seq, ALDH2 had low expression ( p= 3.83E-12, log2Foldchange = 1.158), and ENO1 was up-regulated ( p= 0.0004, log2Foldchange = 1.210 ). ALDH2 is correlated with better survival (HR = 0.47, 95% CI (0.37-0.61), logrankP = 6.5E-10), while ENO1 was inversely associated with better prognosis (HR = 1.67,95% CI (1.32-2.12), logrankP = 1.9E-05) in K-M Plotter. Conclusions: In this study, we discovered new differential expression and prognosis related proteins, ALDH2 and ENO1, and they are worthy of further exploration into the underlying mechanism.
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Arinova, Anar, and Arailym Nurpeissova. "Electrophoretic Deposition of Polyethylene Oxide-Based Gel-Polymer Electrolyte for 3D Lithium-Ion Batteries." ECS Meeting Abstracts MA2023-02, no. 23 (December 22, 2023): 3280. http://dx.doi.org/10.1149/ma2023-02233280mtgabs.
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Despite the advances made in lithium-ion batteries (LIBs) over the last few decades, improved energy and power density is still required for portable devices, transit, and stationary applications [1-3]. Along with developing new materials, optimizing the shape of the battery electrodes is critical for improving the battery performance since the structure of these electrodes has a large impact on ion movement and reaction kinetics. The appeal of 3D structured batteries comes from certain operating features that are not available with conventional 2D geometries. One important feature is the ability to increase areal capacity (mAh cm−2) within a given footprint area of the electrode .Up to today, various methods were employed to coat the foam-type 3D electrodes with polymer or solid-state electrolytes: spin coating, layer-by-layer, and drop coating. Nonetheless, only a number of full-cell operation cases were disclosed, which led to the conclusion that, despite many attempts, drawbacks still exist in solving the homogeneous coating problem of polymer electrolytes. In this work, we report on the simple and facile coating process of polymer electrolyte on the intricate 3D structured NiO@Ni foam anode with the electrophoretic technique. To the best of our knowledge, it was not yet reported in the literature. The conformally coated polymer layer is proven to be very thin and homogeneous without any defects. The NiO@Ni foam/PEO configuration without the use of a commercial separator displayed stable cyclability up to 100 cycles with capacity retention of 88% and coulombic efficiency of 99%. The results are very promising for solving the problem of integrating polymer electrolytes onto any 3D structured anodes. In summary, a facile and simple electrophoretic technique was employed to conformally coat PEO gel-polymer electrolyte onto foam-type NiO/Ni anode for LIBs for the first time. Two electrodes of NiO/Ni foam as a working electrode and Pt metal as a counter electrode were used for the EPD process. Where the PEO solution acted as an electrolyte and, in the end, formed on the NiO/Ni foam surface. Through investigating various layers of PEO, it was concluded that the method allowed obtaining homogeneous thin films of around 2.66 μm for optimal 10 layers, which were free of cracks and defects. The electrochemical performance of the gel-polymer electrolyte PEO with commercially available liquid electrolyte 1M LiPF6 electrolyte solution in EC/EMC/DC was comparable to the traditional separator when used without it in a half-cell with lithium. The cycling stability results displayed an outstanding performance for the PEO electrolyte, delivering a capacity of 406 mAh g-1 at a 0.1 C rate after the 100th cycle. The stability of the formed PEO on the surface of the anode was demonstrated by FTIR analysis after 100 cycles. The stated simple approach allowed a cell operation at room temperature without a commercial separator, which is an excellent result for further developing high-energy-density full 3D batteries.
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Albenzio, Marzia, Antonella Santillo, Francesca d'Angelo, and Agostino Sevi. "Focusing on casein gene cluster and protein profile in Garganica goat milk." Journal of Dairy Research 76, no. 1 (January 5, 2009): 83–89. http://dx.doi.org/10.1017/s0022029908003853.
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A survey was carried out in eight goat dairy farms, a total of 71 individual Garganica goat milk samples were collected for genomic DNA extraction. Casein alleles and haplotype frequencies of Garganica population were estimated. Individual milks were also analysed for chemical composition, rheological properties, and protein profile. The strong A* allele of CSN1S1 was predominant in the population investigated, the weak allele F of CSN1S1 showed a relatively high frequency and the null alleles N and 01 were first observed in this breed. At CSN1S2 locus the strong A* allele was the most frequent, followed by the F allele and the null allele. The strong A* allele was predominant at CSN2 locus, and relatively high incidence of null allele 0 was observed. CSN3 locus was monomorphic for B* allele. The exact test of sample differentiation based on haplotype frequencies discriminate the farms into two groups characterized by the highest frequency of strong (S-CSN1S1) or weak (W-CSN1S1) alleles at CSN1S1. Protein and casein contents were higher in the group characterized by strong allele than in the group with weak allele at CSN1S1. The 2D electrophoresis technique was performed to screen goat casein variability at the protein level and to evaluate global casein genotype (αs1, αs2, β and κ-CN). Gels displayed the protein profile associated with casein genotype, and demonstrated differences in the protein expression deriving from interactions between loci. The variability of goat casein loci in Garganica goat breed could be exploited to differentiate the population on the basis of milk utilization and could represent a strategy to preserve the genotype of this autochthonous breed.
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Xie, Hongdong, Yanyan Tao, Jing Lv, Ping Liu, and Chenghai Liu. "Proteomic Analysis of the Effect of Fuzheng Huayu Recipe on Fibrotic Liver in Rats." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/972863.
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Hepatic fibrosis is a common pathological process of chronic liver diseases and would lead to cirrhosis, and Fuzheng Huayu (FZHY) is an effective Chinese herbal product against liver fibrosis. This study observes FZHY influence on proteome of fibrotic liver with differential proteomic approach and aims to understand FZHY multiple action mechanisms on liver fibrosis. The liver fibrosis models were induced with intraperitoneal injection of dimethylnitrosamine for 4 weeks in rats and divided into model control (model) and FZHY-treated (FZHY) groups, while normal rats were used as normal control (normal). After model establishment, rats in FZHY groups were administered 4 g/kg wt of FZHY for 4 weeks, and normal and model groups were given the same volume of saline. The liver proteins in the above 3 groups were separated by two-dimensional gel electrophoresis (2-DE), the differentially expressed spots were analyzed and compared between normal and model or model and FZHY groups, and then the proteins were identified with mass spectrum analysis and validated partially with western blot and real-time PCR. 1000~1200 spots were displayed on each 2D gel, and a total of 61 protein spots were found with significant intensity difference between normal control or FZHY and model control. 23 most obviously differential spots were excised, and in-gel digestion and 21 peptide mass fingerprints (PMF) were obtained with MALDI-TOF MS analysis, and 14 proteins were identified through protein database searching. Among 14 differentially expressed proteins, 8 proteins in normal and FZHY groups had the same tendency of differential expression compared with the ones in model group. And one of them, vimentin, was validated by western blot and real-time PCR analyses. Our study reveals 12 proteins responsible for fibrogenesis induced by DMN in rats, and among them, 8 proteins in fibrotic liver were regulated by FZHY, including aldehyde dehydrogenase, vimentin isoform (CRA_b), gamma-actin, vimentin, fructose-bisphosphate aldolase B, aldo-keto reductase, S-adenosylhomocysteine hydrolase isoform, and HSP90. It indicates that the action mechanism of FZHY antiliver fibrosis may be associated with modulation of proteins associated with metabolism and stress response, as well as myofibroblast activation. The study provides new insights and data for exploring the liver fibrogenesis pathophysiology and FZHY action mechanism against liver fibrosis.
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Heinrich, Daniel, Marc Weinkauf, Grit Hutter, Kristina Decheva, Yvonne Zimmermann, Wolfgang Hiddemann, and Martin H. Dreyling. "Differential Regulation Patterns of Anti-CD20 Antibodies GA101 and Rituximab in Mantle Cell Lymphoma." Blood 116, no. 21 (November 19, 2010): 1839. http://dx.doi.org/10.1182/blood.v116.21.1839.1839.
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Abstract Abstract 1839 Background: Mantle cell lymphoma (MCL) is a distinct lymphoma subtype characterized by a poor long-term prognosis. Rituximab, a chimeric type I anti-CD20 antibody has shown an anti-proliferative effect in MCL cell lines and is meanwhile widely clinically applied in combination with chemotherapy. GA101, a type II, glycoengineered CD20 IgG1 antibody has been shown to result in higher direct cell death induction and increased ADCC in comparison to rituximab. In previous experiments GA101 displayed a significant higher cytotoxicity in comparison to rituximab. Aim of this study was the elucidation of the involved downstream signal pathways of the two antibodies. Methods: In two sensitive MCL cell lines (Rec-1, Granta-519) we determined the effect of GA101, rituximab and the combination of both antibodies on cell viability and proliferation. Granta 519 and Rec-1 were treated at a cell density of 5×105 cells/ml with GA101 or rituximab at a previously defined dose of 10 μg/ml. After 4h of exposure samples of 3×106 cells were harvested and processed for 2D-PAGE (polyacrylamide gel electrophoresis) analysis. Protein spots with altered expression after antibody treatment from untreated controls were identified and analyzed by mass spectrometry (MALDI-TOF). In parallel, Affymetrix micro-array analysis of MCL cell lines (Granta-519, HBL-2, Jeko-1, Rec-1 and Z-138) was performed after 4h exposure with either rituximab or GA101. To determine downstream pathways, Ingenuity Pathway Analysis of the identified genes was performed. Results: After mono-exposure with GA101 70% and 40 % cell reduction was achieved in Granta-519 and Rec-1, respectively. In contrast, rituximab led to 25% and 5% in Granta-519 and Rec-1. Interestingly, combination of both antibodies resulted in a cytotoxicity comparable to rituximab monotherapy. Computer-based analysis of the respective 2D-PAGE protein maps revealed 40 and 39 distinct differently expressed protein spots after GA101 and rituximab treatment, respectively. 23 of these protein spots were commonly altered after both antibodies (e.g. CCDC158, MACF1, RAB39, RAD23B) whereas after GA101 treatment 17 proteins (e.g. ENO1, MKI67, NPM1, HSPA5) and after rituximab 16 proteins (e.g. DST, G3BP2, LMO7, PSMD13) were uniquely altered. Micro-array analysis resulted in 2–3 (Granta 519) to 14–78 (HBL; GA101 and rituximab respectively) modulated genes after antibody exposure in all five distinct MCL cell lines. Again, applying a fold-change cut-off of 2, unique candidate genes after GA101 (EGR2, EGR3, NFATC1, SPRY2, ZBTB24 (includes EG: 9841)) and rituximab exposure (BCL2A1, CHL1, LILRA4, LPL, LY9, RHEBL1, SOX11, WNT3) were affected in multiple cell lines. Interestingly, transcriptome and proteome-based analysis characterized different sets of candidate molecules, which were however were mapped to common cellular functions including e.g. “cellular growth and proliferation”, “cell death” and “cell cycle”. Combination of both antibodies resulted in a rituximab-like expression pattern, both on RNA and protein level. Conclusions: Our analyses identified different and antibody-specific downstream expression patterns of GA101 and rituximab, which may represent the molecular basis of the superior effect of GA101 in comparison to rituximab. The simultaneous application of both antibodies resulted in a rituximab-like expression pattern of affected cellular functions and canonical pathways. These data will help to identify a molecular-based rationale for future combined therapeutic approaches and avoid potential antagonist effects. Disclosures: Dreyling: Roche: Support of in vitro studies in MCL.
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Szpurka, Hadrian, Andrew E. Schade, Mahrukh K. Ganapathi, and Jaroslaw P. Maciejewski. "Dysfunction of Lipid Raft Microdomains and Defective Apoptotic Signal Transduction in PNH." Blood 108, no. 11 (November 16, 2006): 122. http://dx.doi.org/10.1182/blood.v108.11.122.122.
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Abstract Mutation of PIG-A gene in the hematopoietic stem cell is the primary pathogenic lesion in paroxysmal nocturnal hemoglobinuria (PNH). However, the key element to the understanding of the disease evolution is the ability of the defective hematopoietic clone to outgrow their normal counterparts. It is believed that this growth advantage operates only in the pro-apoptotic milieu that exists in the context of immune-mediated bone marrow failure. The lack of glycosylphosphatidyl inositol-anchored proteins (GPI-AP) on the cell surface of affected cells is likely responsible for the ability of the PNH clone to evade immune attack and expand. GPI-AP are known to be associated with lipid raft microdomains (LRM). LRM contain a large number of receptors and regulatory proteins, whose function depend upon the presence of GPI-AP. Consequently, we hypothesized that GPI-AP deficiency can result in altered LRM which can either blunt proaptoptotic or inhibitory signals or possibly lead to exaggeration of prosurvival stimuli. We utilized paired GPI-AP-deficient (CD−) and wild type (CD+) EBV-transformed B cells derived from a PNH patient, as well as similarly paired lines derived from K562. LRM isolation was performed using sucrose density gradient centrifugation. PNH cells clearly displayed an altered protein repertoire in the LRM, based on analysis by 2D gel electrophoresis and SDS-PAGE, some of which is likely due to the loss of GPI-AP. Immunoblotting revealed lack of the GPI-AP CD55, while Lyn kinase, a known raft-associated protein was present in both PNH and WT LRM. However, an altered LRM function in PNH cells was illustrated via phosphotyrosine immunoblotting: rafts from PNH cell showed a distinct phosphorylation pattern from that observed in WT cells, suggesting distinct raft-dependent signaling in these paired cell lines. In subsequent experiments we have studied the functional consequences of an altered LRM structure in PNH. Two models were established. I) Based on the association of Fas with LRM we stipulated that it may not adequately transduce apoptotic signals in GPI-AP deficient rafts. While Fas was present in LRM of both CD+ and CD− cells as shown by Western blot, in GPI-AP deficient CD− cells, the apoptotic response to Fas agonist CH11 was attenuated in comparison to CD+ cells (23% vs. 60% of apoptotic cells). II) In the second model, we measured differential response of PNH cells to TNFa signaling, shown to be associated with LRM. As a readout, we used TNFa-induced p38 MAPK phosphorylation, which was shown to suppress hematopoiesis; PNH as compared to WT cells showed markedly reduced phosphorylation of p38 after TNF treatment, and exhibited increased basal phosphorylation of NFkB and AKT. These results suggest decreased inhibitory and increased survival signaling in PNH cells treated with TNF. Importantly, we observe increased activity of PDK1, the PI3K-dependent enzyme upstream of AKT, in LRM of PNH cells versus WT cells. Thus, our data show that lipid rafts in PNH cells orchestrate a skewed pro-survival signaling response that may play an important role in their ability to persist in the immune-mediated pro-apoptotic milieu that is a hallmark of bone marrow failure.
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Beycan, Ayhan, Duygu Fatma Ozel Demiralp, Klara Dalva, and Meral Beksaç. "Comparative Proteomic Analysis of Bone Marrow Plasma Cells By Using Mass Spectrometry Based Bottom up Proteomic Strategies." Blood 126, no. 23 (December 3, 2015): 5334. http://dx.doi.org/10.1182/blood.v126.23.5334.5334.
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Abstract Aim : As all other cancer types, multiple myeloma is a complex disease, yet its molecular mechanism that is not fully understood and needs to be furtherly investigated. Besides, the molecular mechanism responsible from the transition of healthy cells into malignant ones is still under investigation. In such comparative studies, human myeloma cell lines are preferred as positive control, while tonsillar plasma cells are used as negative control. The aim of this study is to perform a comparative proteomic analysis regarding the progression of Multiple Myeloma (MM) by using protein profiles of plasma cells obtained from MGUS, SMM, symptomatic myeloma patients, tonsil plasma cells obtained from healthy individuals and human myeloma cells of cell line origin to elucidate the underlying molecular mechanism involved in myeloma. Material and Methods: Five groups were adopted in the experiment. Of these groups, MM patients were classified mainly into three groups according to marrow plasma cell content (PCC) validated by flow cytometry: group1: 0-9, group 2: 10-20 and group 3: >20 %. The fourth group consisted of human myeloma cell line (HMCL) RPMI 8226 and was designated as positive control, while tonsil plasma cells were used as negative control and specified as the fifth group. Marrow samples were collected from 30 patients newly diagnosed with Multiple Myeloma ( n: 28 symptomatic and n: 1 smoldering (SMM)) and MGUS (n: 1)).Tonsil plasma cells were isolated from healthy tonsil tissue treated with trypsin enzyme and were then confirmed by using Selection Kit microbead specific for EasySep Human CD138 marker. Protein profile maps of groups were obtained via 2D gel electrophoresis and comparative analysis and detection of up/down regulated protein spots was performed by using PDQuest 8.01 software. Proteins from differently expressed protein spots were then identified using Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI) by Peptide Mass Fingerprinting (PMF) analysis Results: By using bottom up strategies, nine of the significantly expressed protein spots were identified with PMF analyses. The identified proteins are as follows:, Calreticulin (ERp60), Endoplasmin/ tumor rejection antigen (ERp99), MZB1(Marginal zone B and B1 cell specific protein/ pERp1), Actin cytoplasmic1 (ACTB), Thioredoxin domain-containing protein 5 (TXNDC5/ERp46), Protein disulfide isomerase (PDI) and HLA class I histocompatibility antigen (MHC class I antigen A*2). According to the results, calreticulin (which functions as chaperones and is responsible for Ca+2regulation and cell proliferation) showed a weak positive correlation with increase in patient PC content. The level was lowest in normal B cells. Endoplasmin/ tumor rejection antigen (which is assigned as chaperones and function in apoptotic process and Ca+2 regulation)declined by increase in PC content in patient samples. Level was lowest in HMCL. Marginal zone B and B1 cell specific protein (which functions as chaperones and is involved in apoptotic process, oxidoreductase activity, regulation of Ca+2, cell proliferation and activates B cell quantiity) correlated with PC percentage and was highest in HCML and lowest in normal B cells. Actin cytoplasmic1 protein (which functions in cell proliferation)synthesis showed a minimum decline in synthesis by increase in PC content .Level was highest in normal B cells. Thioredoxin domain-containing protein 5 which plays role in apoptotic process and shows oxireductase activity within MM/MGUS plasma cells also displayed a positive correlation but we were not able to detect a difference in HMCL and normal B cells which were synthesized less than the patient samples. Protein disulfide isomerase (which acts as chaperones and plays part in apoptotic process and shows oxireductase function) has a weak negative correlation with PC content and synthesis was lowest in HMCL. HLA class I histocompatibility antigen production did not correlate with PC content , it was higher in normal B cells compared to HMCL. Conclusion: In conclusion in this study we were able to confirm our previous findings which were presented at ASH 2013 by comparing the results with normal tonsil CD138+ plasma cells and a HMCL. Our study results suggest that the proteins which are involved in Calcium metabolism, chaperone binding and oxidative stress are also associated with low vs. high proliferation profile in myeloma. Disclosures No relevant conflicts of interest to declare.
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De Filippis, Luigi, and Elisabeth Magel. "Identification of biochemical differences between the sapwood and transition zone in Robinia pseudoacacia L. by differential display of proteins." Holzforschung 66, no. 4 (May 1, 2012). http://dx.doi.org/10.1515/hf.2011.178.
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Abstract The predominant proteins and enzymes in the sapwood and transition zones of Robinia pseudoacacia L. were identified and expressed by two methods: 2D SDS-PAGE (two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS). Large differences in the amount of proteins extracted were observed between the bark, sapwood and transition zones. Soluble proteins strongly expressed in sapwood have been identified, and the results interpreted to mean that these proteins are responsible for carbohydrate metabolism and flavonoid turnover. By contrast, proteins strongly expressed in the transition zone are mainly responsible for flavonoid biosynthesis. Lectins were found in protein fractions of both sapwood and the transition zone, and heat-stress proteins were detected only in the transition zone. The results are a further proof that flavonoids are synthesised directly at the transition zone between sapwood and heartwood, and that materials deposited in the sapwood are the source for synthesis of metabolites in heartwood, such as flavonoids and tannins.
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Agnetti, Giulio, Steven T. Elliott, Lesley A. Kane, Christina Yung, Khalid Chakir, Daya Samantapudi, Carlo Guarnieri, Claudio M. Caldarera, David A. Kass, and Jennifer E. Van Eyk. "Abstract 2973: Effects Of Cardiac Resynchronization Therapy On The Mitochondrial Proteome In A Canine Model Of Heart Failure." Circulation 116, suppl_16 (October 16, 2007). http://dx.doi.org/10.1161/circ.116.suppl_16.ii_664-a.
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Cardiac resynchronization therapy (CRT) is a procedure used in the clinics to ameliorate the symptoms associated with heart failure-induced conduction disturbances and ventricular dyssynchrony. The molecular modifications underlying the beneficial effects of CRT have not been completely clarified. Recent investigations suggest that CRT may reduce apoptosis of cardiac myocytes. Mitochondria are likely to be major players in this benign transition due to their role in both energy production and apoptosis regulation. Therefore the proteome of cardiac mitochondria was investigated in a canine model for heart failure alternatively submitted to CRT. Methods and results: Mitochondria-enriched fractions obtained from sections of the left ventricular free wall of dyssynchrony-induced heart failure (DHF) dogs alternatively submitted to CRT were analyzed through two-dimensional gel electrophoreisis (2DE). Roughly 1200 protein spots were visualized on the 2D gels after silver staining. Software-assisted differential display analysis indicated that the density of almost 50 protein spots was significantly changed upon CRT. These spots were selected and identified through mass spectrometry. Among other changes six forms of ATP synthase beta subunit were decreased 2- to 3-fold (0.0001≤p≤0.047), as well as four forms of cytochrome C (2-fold decrease, 0.002≤p≤0.047). The existence of multiple forms of a protein suggests for the presence of post-translational modifications (PTM). Therefore the phosphorylation (a common PTM) status of mitochondrial proteome was also monitored by combining the differential-in-gel electrophoresis (DIGE) technology with alkaline phosphatase teatment. ATP synthase beta subunit was differently phosphorylated in CRT samples comparing to DHF as indicated by the change in its isoelectric point upon treatment with alkaline phosphatase. Conclusions: The proteomic investigation of mitochondria revealed a previously unseen presence of PTM mechanisms at the mitochondria level in heart failure and CRT. Phosphorylation mechanisms in the mitochondria may play a prominent role in modulating cardiac function, as observed for the beneficial effects of CRT.
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Cann, Paul, Malika Chabi, Aliénor Delsart, Chrystelle Le Danvic, Jean-Michel Saliou, Manon Chasles, Matthieu Keller, and Patricia Nagnan-Le Meillour. "The olfactory secretome varies according to season in female sheep and goat." BMC Genomics 20, no. 1 (October 30, 2019). http://dx.doi.org/10.1186/s12864-019-6194-z.
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Abstract Background Small ungulates (sheep and goat) display a seasonal breeding, characterised by two successive periods, sexual activity (SA) and sexual rest (SR). Odours emitted by a sexually active male can reactivate the ovulatory cycle of anoestrus females. The plasticity of the olfactory system under these hormonal changes has never been explored at the peripheral level of odours reception. As it was shown in pig that the olfactory secretome (proteins secreted in the nasal mucus) could be modified under hormonal control, we monitored its composition in females of both species through several reproductive seasons, thanks to a non-invasive sampling of olfactory mucus. For this purpose, two-dimensional gel electrophoresis (2D-E), western-blot with specific antibodies, MALDI-TOF and high-resolution (nano-LC-MS/MS) mass spectrometry, RACE-PCR and molecular modelling were used. Results In both species the olfactory secretome is composed of isoforms of OBP-like proteins, generated by post-translational modifications, as phosphorylation, N-glycosylation and O-GlcNAcylation. Important changes were observed in the olfactory secretome between the sexual rest and the sexual activity periods, characterised in ewe by the specific expression of SAL-like proteins and the emergence of OBPs O-GlcNAcylation. In goat, the differences between SA and SR did not come from new proteins expression, but from different post-translational modifications, the main difference between the SA and SR secretome being the number of isoforms of each protein. Proteomics data are available via ProteomeXchange with identifier PXD014833. Conclusion Despite common behaviour, seasonal breeding, and genetic resources, the two species seem to adapt their olfactory equipment in SA by different modalities: the variation of olfactory secretome in ewe could correspond to a specialization to detect male odours only in SA, whereas in goat the stability of the olfactory secretome could indicate a constant capacity of odours detection suggesting that the hallmark of SA in goat might be the emission of specific odours by the sexually active male. In both species, the olfactory secretome is a phenotype reflecting the physiological status of females, and could be used by breeders to monitor their receptivity to the male effect.
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King, Abigail, Yiwei Zhao, Alexandru Lazar, Margeaux Capron, Niranjan Thiruvur, and Xinrong Liu. "Methods comparison of two‐dimensional gel electrophoresis for host cell protein characterization." Biotechnology Progress, March 18, 2024. http://dx.doi.org/10.1002/btpr.3452.
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AbstractTwo‐dimensional electrophoresis (2DE) is a gel‐based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS‐PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two‐dimensional difference gel electrophoresis (2D‐DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non‐traditional method of 2D‐DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP‐based applications.
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Lu, Fengkun, Wenjing Duan, Yue Cui, Junwei Zhang, Dong Zhu, Ming Zhang, and Yueming Yan. "2D-DIGE based proteome analysis of wheat-Thinopyrum intermedium 7XL/7DS translocation line under drought stress." BMC Genomics 23, no. 1 (May 14, 2022). http://dx.doi.org/10.1186/s12864-022-08599-1.
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Abstract Background Drought stress is the most limiting factor for plant growth and crop production worldwide. As a major cereal crop, wheat is susceptible to drought. Thus, discovering and utilizing drought-tolerant gene resources from related species are highly important for improving wheat drought resistance. In this study, the drought tolerance of wheat Zhongmai 8601-Thinopyrum intermedium 7XL/7DS translocation line YW642 was estimated under drought stress, and then two-dimensional difference gel electrophoresis (2D-DIGE) based proteome analysis of the developing grains was performed to uncover the drought-resistant proteins. Results The results showed that 7XL/7DS translocation possessed a better drought-tolerance compared to Zhongmai 8601. 2D-DIGE identified 146 differential accumulation protein (DAP) spots corresponding to 113 unique proteins during five grain developmental stages of YW642 under drought stress. Among them, 55 DAP spots corresponding to 48 unique proteins displayed an upregulated expression, which were mainly involved in stress/defense, energy metabolism, starch metabolism, protein metabolism/folding and transport. The cis-acting element analysis revealed that abundant stress-related elements were present in the promoter regions of the drought-responsive protein genes, which could play important roles in drought defense. RNA-seq and RT-qPCR analyses revealed that some regulated DAP genes also showed a high expression level in response to drought stress. Conclusions Our results indicated that Wheat-Th. intermedium 7XL/7DS translocation line carried abundant drought-resistant proteins that had potential application values for wheat drought tolerance improvement.
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Lazensky, Rebecca, Cecilia Silva-Sanchez, Kevin J. Kroll, Marjorie Chow, Sixue Chen, Katie Tripp, Michael T. Walsh, and Nancy D. Denslow. "Investigating an increase in Florida manatee mortalities using a proteomic approach." Scientific Reports 11, no. 1 (February 19, 2021). http://dx.doi.org/10.1038/s41598-021-83687-y.
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AbstractTwo large-scale Florida manatee (Trichechus manatus latirostris) mortality episodes were reported on separate coasts of Florida in 2013. The east coast mortality episode was associated with an unknown etiology in the Indian River Lagoon (IRL). The west coast mortality episode was attributed to a persistent Karenia brevis algal bloom or ‘red tide’ centered in Southwest Florida. Manatees from the IRL also had signs of cold stress. To investigate these two mortality episodes, two proteomic experiments were performed, using two-dimensional difference in gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantification (iTRAQ) LC–MS/MS. Manatees from the IRL displayed increased levels of several proteins in their serum samples compared to controls, including kininogen-1 isoform 1, alpha-1-microglobulin/bikunen precursor, histidine-rich glycoprotein, properdin, and complement C4-A isoform 1. In the red tide group, the following proteins were increased: ceruloplasmin, pyruvate kinase isozymes M1/M2 isoform 3, angiotensinogen, complement C4-A isoform 1, and complement C3. These proteins are associated with acute-phase response, amyloid formation and accumulation, copper and iron homeostasis, the complement cascade pathway, and other important cellular functions. The increased level of complement C4 protein observed in the red tide group was confirmed through the use of Western Blot.
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Urban, Milan O., Sébastien Planchon, Irena Hoštičková, Radomira Vanková, Peter Dobrev, Jenny Renaut, Miroslav Klíma, and Pavel Vítámvás. "The Resistance of Oilseed Rape Microspore-Derived Embryos to Osmotic Stress Is Associated With the Accumulation of Energy Metabolism Proteins, Redox Homeostasis, Higher Abscisic Acid, and Cytokinin Contents." Frontiers in Plant Science 12 (June 11, 2021). http://dx.doi.org/10.3389/fpls.2021.628167.
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The present study aims to investigate the response of rapeseed microspore-derived embryos (MDE) to osmotic stress at the proteome level. The PEG-induced osmotic stress was studied in the cotyledonary stage of MDE of two genotypes: Cadeli (D) and Viking (V), previously reported to exhibit contrasting leaf proteome responses under drought. Two-dimensional difference gel electrophoresis (2D-DIGE) revealed 156 representative protein spots that have been selected for MALDI-TOF/TOF analysis. Sixty-three proteins have been successfully identified and divided into eight functional groups. Data are available via ProteomeXchange with identifier PXD024552. Eight selected protein accumulation trends were compared with real-time quantitative PCR (RT-qPCR). Biomass accumulation in treated D was significantly higher (3-fold) than in V, which indicates D is resistant to osmotic stress. Cultivar D displayed resistance strategy by the accumulation of proteins in energy metabolism, redox homeostasis, protein destination, and signaling functional groups, high ABA, and active cytokinins (CKs) contents. In contrast, the V protein profile displayed high requirements of energy and nutrients with a significant number of stress-related proteins and cell structure changes accompanied by quick downregulation of active CKs, as well as salicylic and jasmonic acids. Genes that were suitable for gene-targeting showed significantly higher expression in treated samples and were identified as phospholipase D alpha, peroxiredoxin antioxidant, and lactoylglutathione lyase. The MDE proteome profile has been compared with the leaf proteome evaluated in our previous study. Different mechanisms to cope with osmotic stress were revealed between the genotypes studied. This proteomic study is the first step to validate MDE as a suitable model for follow-up research on the characterization of new crossings and can be used for preselection of resistant genotypes.
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Laffoon, Scott B., James D. Doecke, Anne M. Roberts, Jennifer A. Vance, Benjamin D. Reeves, Kelly K. Pertile, Rebecca L. Rumble, et al. "Analysis of plasma proteins using 2D gels and novel fluorescent probes: in search of blood based biomarkers for Alzheimer’s disease." Proteome Science 20, no. 1 (January 26, 2022). http://dx.doi.org/10.1186/s12953-021-00185-9.
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Abstract Background The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer’s disease. Methods The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. Results We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. Conclusions This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.
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Teixeira, Priscila Camillo, Axel Ducret, Hanno Langen, Everson Nogoceke, Ronaldo Honorato Barros Santos, João Paulo Silva Nunes, Luiz Benvenuti, et al. "Impairment of Multiple Mitochondrial Energy Metabolism Pathways in the Heart of Chagas Disease Cardiomyopathy Patients." Frontiers in Immunology 12 (November 12, 2021). http://dx.doi.org/10.3389/fimmu.2021.755782.
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Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy occurring in 30% of the 6 million infected with the protozoan Trypanosoma cruzi in Latin America. Survival is significantly lower in CCC than ischemic (IC) and idiopathic dilated cardiomyopathy (DCM). Previous studies disclosed a selective decrease in mitochondrial ATP synthase alpha expression and creatine kinase activity in CCC myocardium as compared to IDC and IC, as well as decreased in vivo myocardial ATP production. Aiming to identify additional constraints in energy metabolism specific to CCC, we performed a proteomic study in myocardial tissue samples from CCC, IC and DCM obtained at transplantation, in comparison with control myocardial tissue samples from organ donors. Left ventricle free wall myocardial samples were subject to two-dimensional electrophoresis with fluorescent labeling (2D-DIGE) and protein identification by mass spectrometry. We found altered expression of proteins related to mitochondrial energy metabolism, cardiac remodeling, and oxidative stress in the 3 patient groups. Pathways analysis of proteins differentially expressed in CCC disclosed mitochondrial dysfunction, fatty acid metabolism and transmembrane potential of mitochondria. CCC patients’ myocardium displayed reduced expression of 22 mitochondrial proteins belonging to energy metabolism pathways, as compared to 17 in DCM and 3 in IC. Significantly, 6 beta-oxidation enzymes were reduced in CCC, while only 2 of them were down-regulated in DCM and 1 in IC. We also observed that the cytokine IFN-gamma, previously described with increased levels in CCC, reduces mitochondrial membrane potential in cardiomyocytes. Results suggest a major reduction of mitochondrial energy metabolism and mitochondrial dysfunction in CCC myocardium which may be in part linked to IFN-gamma. This may partially explain the worse prognosis of CCC as compared to DCM or IC.