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Gel Analysis (gel + analysis)
Selected AbstractsSecretome analysis of differentially induced proteins in rice suspension-cultured cells triggered by rice blast fungus and elicitorPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2009Sun Tae Kim Abstract Secreted proteins were investigated in rice suspension-cultured cells treated with rice blast fungus Magnaporthe grisea and its elicitor using biochemical and 2-DE coupled with MS analyses followed by their in planta mRNA expression analysis. M. grisea and elicitor successfully interacted with suspension-cultured cells and prepared secreted proteins from these cultures were essentially intracellular proteins free. Comparative 2-D gel analyses identified 21 differential protein spots due to M. grisea and/or elicitor over control. MALDI-TOF-MS and ,LC-ESI-MS/MS analyses of these protein spots revealed that most of assigned proteins were involved in defense such as nine chitinases, two germin A/oxalate oxidases, five domain unknown function 26 (DUF 26) secretory proteins, and ,-expansin. One chitin binding chitinase protein was isolated using chitin binding beads and strong enzymatic activity was identified in an in-gel assay. Interestingly, their protein abundance correlated well at transcript levels in elicitor-treated cultures as judged by semi-quantitative RT-PCR. Each identified differentially expressed protein group was compared at transcript levels in rice leaves inoculated with incompatible (KJ401) and compatible (KJ301) races of M. grisea. Time-course profiling revealed their inductions were stronger and earlier in incompatible than compatible interactions. Identified secreted proteins and their expression correlation at transcript level in suspension-cultured cells and also in planta suggest that suspension-cultured cells can be useful to investigate the secretome of rice blast,pathogen interactions. [source] Proteomic analysis of liver cancer cells treated with 5-Aza-2,-deoxycytidine (AZA),DRUG DEVELOPMENT RESEARCH, Issue 1 2009Shujun Bai Abstract 5-Aza-2,-deoxycytidine (AZA) is a potent inhibitor of DNA methylation that exhibits anti-tumor activity in a variety of tumor cells via reactivation of tumor suppressor genes. However, few studies have been done on the biological and clinical significance of AZA in human hepatocellular carcinoma. To identify potential genes that may be aberrantly methylated and confer growth advantage to neoplastic cells and to better understand the molecular mechanism(s) underlying AZA anti-tumor activity, a proteomics approach was used to annotate global gene expression changes of HepG2 cell line pre- and post-treatment with AZA. A total of 56 differentially expressed proteins were identified by 2D gel analysis, 48 of which were up-regulated while the remaining 8 were down regulated. Among the identified proteins, eight of these showed marked changed proteins, including seven up-regulated proteins: glutathione S-transferase P, protein DJ-1, peroxiredoxin-2, UMP-CMP kinase, cytochrome c-type heme lyase, enhancer of rudimentary homolog, profilin-1, and one down-regulated protein, heat-shock protein ,,1. The possible implication of these proteins in hepatocarcinogenesis is discussed. We tested two up-regulated proteins, glutathione S-transferase P and peroxiredoxin-2, using RT-PCR and their expression was consistent with the results obtained in the protein level. Both of these genes were methylated when methylation-specific PCR was used against their promoter regions. Following treatment with AZA, the gene promoter regions were found to be unmethylated, concomitant with overexpression of the proteins compared to HepG2 cells without treatment. These data provide useful information in evaluating the therapeutic potential of AZA for the treatment of HCC. Drug Dev Res 69, 2009. © 2009 Wiley-Liss, Inc. [source] Two-dimensional gel analysis of stress proteins identified in Chironomus flaviplumus (Diptera: Chironomidae) exposed to 4-nonylphenolENTOMOLOGICAL RESEARCH, Issue 3 2010Myoung Chul KIM Abstract Toxicity of 4-nonylphenol (4-NP) in the Chironomidae Chironomus flaviplumus was analyzed using a proteomics approach that involved identifying proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Proteome analysis of 4-NP-treated samples on silver stained gels found alterations in the expression levels of three proteins compared with control samples. Hsp70 proteins, so-called stress proteins, were studied in Chironomus flaviplumus exposed to different concentrations of 4-nonylphenol (0, 30, 100, 150, 300 and 600 mg/L) in the laboratory and in the field in captured animals from site 1 (1 km from a chemical factory) and site 3 (16 km from a chemical factory). Hsp70 proteins were found in all samples tested, including controls, but differed in their expression levels. At more polluted sites (site 1), the samples treated with higher concentrations of 4-NP more strongly expressed Hsp70. 2-D spots were induced or enhanced in gels following injection of 4-NP. Therefore, the induction of stress protein expression in Chironomus flaviplumus, in particular Hsp70, can be used as a biomarker for the evaluation of environmental conditions. [source] Fungal rDNA signatures in coronary atherosclerotic plaquesENVIRONMENTAL MICROBIOLOGY, Issue 12 2007Stephan J. Ott Summary Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology. [source] Trehalose and trehalose-hydrolyzing enzyme in the haemolymph of Locusta migratoria infected with Metarhizium anisopliae strain CQMa102INSECT SCIENCE, Issue 4 2007HUA ZHAO Abstract Topical application of the Metarhizium anisopliae var. acridum specialist strain CQMa102 to the locust Locusta migratoria manilensis results in changes of the concentrations of trehalose and glucose in the haemolymph. Micrographs of the locust haemolymph shows Metarhizium anisopliae can effectivly penetrate the external skeleton of locust and after 2 days infection, the hyphae body will appear in the haemolymph of infected insects. The time in decrease of trehalose concentration coincided with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. Overlay gel analysis indicated there was considerably more trehalose-hydrolysing activity in the haemolymph of locusts infected by fungus than in controls. A comparable isoform was identified in in vitro culture of the fungus, suggesting a fungal origin for the in vivo enzyme. Haemolymph trehalose decreased significantly during mycosis of locusts by M. anisopliae. All these results suggested that this fungus may take advantage of competing nutrient utilization against the insect by its trehalose-hydrolyzing enzyme secretion. It may provide fundamental knowledge for fungal pathogenesis. [source] Rapid detection of bordetella pertussis by real-time PCR using SYBR green I and a LightCycler instrumentJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2004S. K. Poddar Abstract A polymerase chain reaction (PCR) assay in real-time for detection of B. pertussis using SYBR green I as the reporter fluorophore and LightCycler instrument (a thermocycler coupled to a fluorescence detection device) was established and evaluated. The amplified amplicon using series diluted control prototype strain (ATCC strain #9797) of B. pertussis was analyzed for the fluorescent melting profile, and melting temperature (Tm) was determined. When examined, amplicons using a representative set of clinical isolates of B. pertussis were found to have the same Tm value (86 ± 0.5°C, the specificity parameter of detection) as the control prototype strain as expected. Amplified product was also analyzed and detected by agarose gel electrophoresis. The detection limit by fluorescent profile and Tm analysis was 10-fold better than that detected by agarose gel analysis. J. Clin. Lab. Anal. 18:265,270, 2004. © 2004 Wiley-Liss, Inc. [source] PROTEIN PROFILE CHANGES IN ACID ADAPTED LISTERIA MONOCYTOGENES EXHIBITING CROSS-PROTECTION AGAINST AN ACTIVATED LACTOPEROXIDASE SYSTEM IN TRYPTIC SOY BROTHJOURNAL OF FOOD SAFETY, Issue 1 2000SADHANA RAVISHANKAR ABSTRACT Foodborne pathogens often tolerate and survive environmental stress conditions including extreme acidity to varying degrees. One possible reason for this survival may be the production of protective stress proteins during acid shock (ASR) and/or tolerance (ATR) responses. The ASR and ATR of Listeria monocytogenes strains V7, V37 and CA in tryptic soy broth without dextrose acidified with lactic acid were studied. Possible cross-protection of acid adapted cells against an activated lactoperoxidase system was also determined. The strains were either directly challenged at pH 4.0 and 3.5 to study their ASR or initially adapted at pH 5.5 for the equivalent of 1 generation before challenging at pH 4.0 and 3.5 to study their ATR. Adapted and nonadapted cells were challenged at pH 4.5 with or without an activated lactoperoxidase system. In all cases viability was determined by enumeration over a period of 24 or 48 h after challenge and the production of stress proteins analyzed by 2-dimensional gel electrophoresis. While there were some differences in the survival responses for each strain, the acid adapted cells of each strain survived to a greater degree than nonadapted cells at both pH 4.0 (at least 10 fold at 24 h) and pH 3.5 (at least 1000 fold at 6 h) but not at pH 4.5. The acid adapted cells exposed to the lactoperoxidase system survived better (at least 5-fold) than their nonadapted counterparts for all 3 strains at 24 and 48 h. The 2-dimensional gel analysis for all 3 strains showed that the adapted and nonadapted cells underwent a change in their physiology, (at pH 4.0 compared to the control at pH 7.0; at pH 4.5 with the addition of lactoperoxidase system components) in that there was induction as well as repression of several proteins. [source] Photoinactivation of Sindbis Virus Infectivity Without Inhibition of Membrane FusionPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2009Wor Thongthai Photoinactivation of enveloped viruses is commonly associated with damage to fusion proteins and inhibition of membrane fusion capacity. Here we show that photobleaching of Sindbis virus labeled with the membrane localized dye, R18 (octadecyl rhodamine B) causes a dramatic loss of infectivity without observable changes in low-pH triggered membrane fusion to liposomes. Sindbis labeled with DiI (1,1,-dioctadecyl-3,3,3,,3,-tetramethylindocarbocyanine perchlorate) also maintains low-pH triggered membrane fusion capacity, but in contrast to R18, extensive photobleaching of DiI-labeled virus has little effect on infectivity. Electrophoretic gel analysis suggests no cross-linking of viral fusion proteins following photobleaching of dye-labeled Sindbis. These observations have implications for live-cell, single particle tracking studies of dye-labeled Sindbis virus. Our observations suggest that R18 and DiI have different propensities for spontaneous flip-flop in lipid bilayers. [source] Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signaturePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2009Stephane Roche Abstract Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature. [source] Shotgun proteomic analysis of human-induced sputumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2006Ben Nicholas Dr. Abstract Induced sputum is a readily accessible biological fluid whose composition may alter as a consequence of disease. To date, however, the proteins that routinely populate this biofluid are largely unknown, in part due to the technical difficulties in processing such mucin-rich samples. To provide a catalogue of sputum proteins, we have surveyed the proteome of human-induced sputum (sputome). A combination of 2-D gel analysis and GeLC-MS/MS allowed a total of 191 human proteins to be confidently assigned. In addition to the expected components, several hitherto unreported proteins were found to be present, including three members of the annexin family, kallikreins 1 and 11, and peroxiredoxins 1, 2 and 5. Other sets of proteins identified included four proteins previously annotated as hypothetical or conserved hypothetical. Taken together, these data represent the first extensive survey of the proteome of induced sputum and provide a platform for future identification of biomarkers of lung disease. [source] Shotgun proteomic analysis of Chlamydia trachomatisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2005Paul Skipp Abstract Chlamydiae are widespread bacterial pathogens responsible for a broad range of diseases, including sexually transmitted infections, pneumonia and trachoma. To validate the existence of hitherto hypothetical proteins predicted from recent chlamydial genome sequencing projects and to examine the patterns of expression of key components at the protein level, we have surveyed the expressed proteome of Chlamydia trachomatis strain,L2. A combination of two-dimensional gel analysis, multi-dimensional protein identification (MudPIT) and nanocapillary liquid chromatography-tandem mass spectrometry allowed a total of 328,chlamydial proteins to be unambiguously assigned. Proteins identified as being expressed in the metabolically inert form, elementary body, of Chlamydia include the entire set of predicted glycolytic enzymes, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. An enzyme central to cell wall biosynthesis was also detected in the intracellular form, reticulate body, of Chlamydia, suggesting that the peptidoglycan is produced during growth within host cells. Other sets of proteins identified include 17 outer membrane-associated proteins of potential significance in vaccine studies and 67,proteins previously annotated as hypothetical or conserved hypothetical. Taken together, ,35% of the predicted proteome for C.,trachomatis has been experimentally verified, representing the most extensive survey of any chlamydial proteome to date. [source] Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administrationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Chiara Piubelli Abstract In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36,spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine. [source] Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systemsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Melanie Y. White Abstract Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein. [source] | ||||||||||||||||