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Sample records for activity-based protein profiling

  1. Activity-Based Protein Profiling of Microbes

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  2. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621. ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity -based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 3.088, year: 2014

  3. Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

    Yinliang Yang

    2013-10-01

    Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

  4. Activity based protein profiling to detect serine hydrolase alterations in virus infected cells

    MdShahiduzzaman; KevinM.Coombs

    2012-01-01

    Activity-based protein profiling (ABPP) is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used rec...

  5. Activity based protein profiling to detect serine hydrolase alterations in virus infected cells

    MdShahiduzzaman

    2012-08-01

    Full Text Available Activity based protein profiling (ABPP is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation and reproduction. ABPP has been used recently to investigate host-virus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of host-virus interaction.

  6. Activity-based protein profiling of ammonia monooxygenase in Nitrosomonas europaea.

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.

    2016-01-29

    Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2-) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). Many alkynes are mechanism-based inactivators of AMO and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with AlexaFluor 647-azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, solubilized and analyzed by SDS-PAGE and IR scanning. A fluorescent 28 kDa polypeptide was observed for cells previously exposed to 17OD, but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD, or for cells not previously exposed to 17OD. The 28 kDa polypeptide was membrane-associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent 28 kDa polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested and amino acid sequences of the peptide fragments were determined by LC-MS analysis. Peptide fragments from amoA were the predominant peptides detected in 17OD-treated samples. In gel digestion and MALDI-TOF/TOF analysis also confirmed the fluorescent 28 kDa polypeptide was amoA.

  7. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

    Bennett, Kristen; Sadler, Natalie C; Wright, Aaron T; Yeager, Chris; Hyman, Michael R

    2016-04-01

    Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

  8. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  9. Disparate Proteome Responses of Pathogenic and Non-pathogenic Aspergilli to Human Serum Measured by Activity-Based Protein Profiling (ABPP)

    Wiedner, Susan D.; Ansong, Charles; Webb-Robertson, Bobbie-Jo M.; Pederson, Leeanna M.; Fortuin, Suereta; Hofstad, Beth A.; Shukla, Anil K.; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

    2013-07-01

    Aspergillus fumigatus is the primary pathogen causing the devastating pulmonary disease Invasive Aspergillosis in immunocompromised individuals. Genomic analysis shows high synteny between A. fumigatus and closely related rarely pathogenic Neosartorya fischeri and Aspergillus clavatus genomes. To investigate the presence of unique or highly inducible protein reactivity in the pathogen, we applied activity-based protein profiling to compare protein reactivity of all three fungi over time in minimal media growth and in response to human serum. We found 350 probe-reactive proteins exclusive to A. fumigatus, including known virulence associated proteins, and 13 proteins associated with stress response exclusive to A. fumigatus culture in serum. Though the fungi are highly orthologous, A. fumigatus has significantly more activity across varied biological process. Only 50% of expected orthologs of measured A. fumigatus reactive proteins were observed in N. fischeri and A. clavatus. Human serum induced processes uniquely or significantly represented in A. fumigatus include actin organization and assembly, transport, and fatty acid, cell membrane, and cell wall synthesis. Additionally, signaling proteins regulating vegetative growth, conidiation, and cell wall integrity, required for appropriate cellular response to external stimuli, had higher reactivity over time in A. fumigatus and N. fisheri, but not in A. clavatus. Together, we show that measured proteins and physiological processes identified solely or significantly over-represented in A. fumigatus reveal a unique adaptive response to human protein not found in closely related, but rarely aspergilli. These unique protein reactivity responses may reveal how A. fumigatus initiates pulmonary invasion leading to Invasive Aspergillosis.

  10. Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

    Sadler, Natalie C.; Angel, Thomas E.; Lewis, Michael P.; Pederson, Leeanna M.; Chauvigne-Hines, Lacie M.; Wiedner, Susan D.; Zink, Erika M.; Smith, Richard D.; Wright, Aaron T.

    2012-10-24

    High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD or if the mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar or elevated relative to standard diet (SD) mice, thereby IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

  11. Direct and two-step activity-based profiling of proteases and glycosidases

    Willems, Lianne Irene

    2014-01-01

    Activity-based protein profiling provides a powerful approach for the monitoring of active enzyme populations in complex biological samples by making use of activity-based probes (ABPs), chemical probes that are designed to bind specifically to the active site of an enzyme (family). The research described in this thesis concerns two main topics. First, new techniques are developed for the two-step labeling of enzymatic activity, a strategy that involves the targeting of enzymes with an ABP fo...

  12. Fluorescent profiling of modular biosynthetic enzymes by complementary metabolic and activity based probes.

    Meier, Jordan L; Mercer, Andrew C; Burkart, Michael D

    2008-04-23

    The study of the enzymes responsible for natural product biosynthesis has proven a valuable source of new enzymatic activities and been applied to a number of biotechnology applications. Protein profiling could prove highly complementary to genetics based approaches by allowing us to understand the activity, transcriptional control, and post-translational modification of these enzymes in their native and dynamic proteomic environments. Here we present a method for the fluorescent profiling of PKS, NRPS, and FAS multidomain modular synthases in their whole proteomes using complementary metabolic and activity based probes. After first examining the reactivity of these activity based probes with a variety of purified recombinant PKS, NRPS, and FAS enzymes in vitro, we apply this duel labeling strategy to the analysis of modular synthases in a human breast cancer cell line and two strains of the natural product producer Bacillus subtilis. Collectively, these studies demonstrate that complementary protein profiling approaches can prove highly useful in the identification and assignment of inhibitor specificity and domain structure of these modular biosynthetic enzymes. PMID:18376827

  13. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  14. Substrate-competitive activity-based profiling of ester prodrug activating enzymes

    Xu, Hao; Majmudar, Jaimeen D.; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H.; Carlson, Heather A.; Showalter, Hollis D.; Martin, Brent R.; Amidon, Gordon L.

    2015-01-01

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a 4-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse, but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design and preclinical

  15. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    Ismail, Hanafy M.; O' Neill, Paul M.; Hong, David; Finn, Robert; Henderson, Colin; Wright, Aaron T.; Cravatt, Benjamin; Hemingway, Janet; Paine, Mark J.

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the target tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.

  16. Protein profiling of cerebrospinal fluid

    Simonsen, Anja H

    2012-01-01

    The cerebrospinal fluid (CSF) perfuses the brain and spinal cord. CSF contains proteins and peptides important for brain physiology and potentially also relevant for brain pathology. Hence, CSF is the perfect source to search for new biomarkers to improve diagnosis of neurological diseases as well...... as to monitor the performance of disease-modifying drugs. This chapter presents methods for SELDI-TOF profiling of CSF as well as useful advice regarding pre-analytical factors to be considered....

  17. Prediction of Protein-Protein Interactions Using Protein Signature Profiling

    Mahmood; A.; Mahdavi; Yen-Han; Lin

    2007-01-01

    Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain inter- actions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as "interacting". In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis ele- gans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area un- der the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs in- creased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on aver- age 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

  18. Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.

    McLellan, Lauren; Forder, Cassie; Cranston, Aaron; Harrigan, Jeanine; Jacq, Xavier

    2016-01-01

    The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors. PMID:27613053

  19. Serum Protein Profile Alterations in Hemodialysis Patients

    Murphy, G A; Davies, R W; Choi, M W; Perkins, J; Turteltaub, K W; McCutchen-Maloney, S L; Langlois, R G; Curzi, M P; Trebes, J E; Fitch, J P; Dalmasso, E A; Colston, B W; Ying, Y; Chromy, B A

    2003-11-18

    Background: Serum protein profiling patterns can reflect the pathological state of a patient and therefore may be useful for clinical diagnostics. Here, we present results from a pilot study of proteomic expression patterns in hemodialysis patients designed to evaluate the range of serum proteomic alterations in this population. Methods: Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOFMS) was used to analyze serum obtained from patients on periodic hemodialysis treatment and healthy controls. Serum samples from patients and controls were first fractionated into six eluants on a strong anion exchange column, followed by application to four array chemistries representing cation exchange, anion exchange, metal affinity and hydrophobic surfaces. A total of 144 SELDI-TOF-MS spectra were obtained from each serum sample. Results: The overall profiles of the patient and control samples were consistent and reproducible. However, 30 well-defined protein differences were observed; 15 proteins were elevated and 15 were decreased in patients compared to controls. Serum from one patient exhibited novel protein peaks suggesting possible additional changes due to a secondary disease process. Conclusion: SELDI-TOF-MS demonstrated dramatic serum protein profile differences between patients and controls. Similarity in protein profiles among dialysis patients suggests that patient physiological responses to end-stage renal disease and/or dialysis therapy have a major effect on serum protein profiles.

  20. Detection of distant evolutionary relationships between protein families using theory of sequence profile-profile comparison

    Venclovas Česlovas

    2010-02-01

    Full Text Available Abstract Background Detection of common evolutionary origin (homology is a primary means of inferring protein structure and function. At present, comparison of protein families represented as sequence profiles is arguably the most effective homology detection strategy. However, finding the best way to represent evolutionary information of a protein sequence family in the profile, to compare profiles and to estimate the biological significance of such comparisons, remains an active area of research. Results Here, we present a new homology detection method based on sequence profile-profile comparison. The method has a number of new features including position-dependent gap penalties and a global score system. Position-dependent gap penalties provide a more biologically relevant way to represent and align protein families as sequence profiles. The global score system enables an analytical solution of the statistical parameters needed to estimate the statistical significance of profile-profile similarities. The new method, together with other state-of-the-art profile-based methods (HHsearch, COMPASS and PSI-BLAST, is benchmarked in all-against-all comparison of a challenging set of SCOP domains that share at most 20% sequence identity. For benchmarking, we use a reference ("gold standard" free model-based evaluation framework. Evaluation results show that at the level of protein domains our method compares favorably to all other tested methods. We also provide examples of the new method outperforming structure-based similarity detection and alignment. The implementation of the new method both as a standalone software package and as a web server is available at http://www.ibt.lt/bioinformatics/coma. Conclusion Due to a number of developments, the new profile-profile comparison method shows an improved ability to match distantly related protein domains. Therefore, the method should be useful for annotation and homology modeling of uncharacterized

  1. Protein Profiling of Preeclampsia Placental Tissues

    Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; JIANG, JING; Lee, Peng; Zhang, David Y.; He, Jin

    2014-01-01

    Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expres...

  2. Investigating homology between proteins using energetic profiles.

    James O Wrabl

    2010-03-01

    Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved

  3. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interac-tion data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automati-cally selects the most appropriate functional classes as specific as possible during the learning proc-ess, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to “biology process” by three measures particularly designed for functional classes organ-ized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  4. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    GAO Lei; LI Xia; GUO Zheng; ZHU MingZhu; LI YanHui; RAO ShaoQi

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interaction data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automatically selects the most appropriate functional classes as specific as possible during the learning process, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to "biology process" by three measures particularly designed for functional classes organized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  5. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles

    Zhong, Cuncong; Yooseph, Shibu

    2016-01-01

    Analyses of metagenome data (MG) and metatranscriptome data (MT) are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM) framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR) gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE) genes. Finally, HMM-GRASPx was used to

  6. DETERMINATION O F TOTAL CELL PROTEIN PROFILES OF Streptomyces SPECIES

    Özdemir K

    2013-07-01

    Full Text Available Present study has been conducted for finding out the total protein profile of bacterial strain Streptomyces sps by sodium dodecyl sulphate polyacrylamide gelelectrophoresis. Total 139 isolates of Streptomyces have been isolated from the soil. Amongst all isolated strain, total 20 isolates were used for getting protein profile by SDS PAGE. Amongst all isolates, 20 isolates were selected for protein profiling and these were divided in two groups. Two strains of Streptomyces i.e. S. violaceus and S. albidoflavus were selected as a reference strain for both groups. Band profile were analyzed and assessed by computer added program BioRad Quantity with the use of Unweighted Pair Group Method of Analysis (UPGMA. As a result o f this computer assisted numeric analysis study, approximately 40 different types of protein bands were reported between 10 or 100 kD molecular weight. Analysis of acquired dendogram on the basis of similarities ratios, all 40 proteins can be divid ed in to 7 groups. In addition, the isolates A4B3G, D145B, S5036.6 and reference isolate S. violaceus were available in the same group, while 805A, C804B, F1705 isolates and reference sample S.albidoflavus were detected in the same group. The test organisms which were similar to each other in terms of morphological and biochemical characters delivered the same protein bands. SDS-PAGE method is an effective method interms of determining taxonomical relations between the various species of genus Streptomyces.

  7. Analysis of protein profiles using fuzzy clustering methods

    Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya; Kartha, V.B; C, Santhosh

    The tissue protein profiles of healthy volunteers and volunteers with cervical cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence  technique  (HPLC-LIF)  developed  in  our  lab.      We analyzed      the protein profile data using different...... clustering methods for their classification followed by various validation  measures.    The  clustering  algorithms  used  for  the  study  were  K-  means,  K- medoid, Fuzzy C-means, Gustafson-Kessel, and Gath-Geva.  The results presented in this study  conclude  that  the  protein  profiles  of  tissue...

  8. Protein Profile of Exosomes from Trabecular Meshwork Cells

    Stamer, WD; Hoffman, EA; Luther, JM; Hachey, DL; Schey, KL

    2011-01-01

    To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were co...

  9. Proteomics of protein expression profiling in tissues with different radiosensitivity

    Ionizing radiation activates multiple signaling pathways, resulting in diverse stress responses including apoptosis, cell cycle arrest, and gene induction. Liver tissue is known to be rather resistant to radiation while a spleen tissue is highly radiosentitive. Our purpose was to compare radioresponse in liver and spleen following exposure to radiation to further investigate the differentially protein expression profile in radiosensitive and radioresistant tissues

  10. Interaction profile-based protein classification of death domain

    Pio Frederic

    2004-06-01

    Full Text Available Abstract Background The increasing number of protein sequences and 3D structure obtained from genomic initiatives is leading many of us to focus on proteomics, and to dedicate our experimental and computational efforts on the creation and analysis of information derived from 3D structure. In particular, the high-throughput generation of protein-protein interaction data from a few organisms makes such an approach very important towards understanding the molecular recognition that make-up the entire protein-protein interaction network. Since the generation of sequences, and experimental protein-protein interactions increases faster than the 3D structure determination of protein complexes, there is tremendous interest in developing in silico methods that generate such structure for prediction and classification purposes. In this study we focused on classifying protein family members based on their protein-protein interaction distinctiveness. Structure-based classification of protein-protein interfaces has been described initially by Ponstingl et al. 1 and more recently by Valdar et al. 2 and Mintseris et al. 3, from complex structures that have been solved experimentally. However, little has been done on protein classification based on the prediction of protein-protein complexes obtained from homology modeling and docking simulation. Results We have developed an in silico classification system entitled HODOCO (Homology modeling, Docking and Classification Oracle, in which protein Residue Potential Interaction Profiles (RPIPS are used to summarize protein-protein interaction characteristics. This system applied to a dataset of 64 proteins of the death domain superfamily was used to classify each member into its proper subfamily. Two classification methods were attempted, heuristic and support vector machine learning. Both methods were tested with a 5-fold cross-validation. The heuristic approach yielded a 61% average accuracy, while the machine

  11. Comparison of major protein antigens and protein profiles of Treponema pallidum and Treponema pertenue.

    Thornburg, R W; Baseman, J B

    1983-01-01

    The protein profiles of Treponema pallidum and Treponema pertenue, the causative agents of syphilis and yaws, respectively, were compared by one- and two-dimensional gel electrophoresis. One-dimensional gels showed essentially no differences in the protein patterns of these treponemes. On two-dimensional gels most radiolabeled protein species were shared; however, variations were noticed in several minor protein species. Antigenic comparison by radioimmunoprecipitation and Western blotting al...

  12. Protein Profile Changes during Porcine Oocyte Aging and Effects of Caffeine on Protein Expression Patterns

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 1...

  13. Bacterial characterization using protein profiling in a microchip separations platform.

    Pizarro, Shelly A; Lane, Pamela; Lane, Todd W; Cruz, Evelyn; Haroldsen, Brent; VanderNoot, Victoria A

    2007-12-01

    A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip gel electrophoresis (CGE) coupled with LIF detection was used to analyze lysates from different bacterial cell lines to obtain signature profiles of the soluble protein composition. The study includes Escherichia coli, Bacillus subtilis, and Bacillus anthracis (Delta Sterne strain) vegetative cells as well as endospores formed from the latter two species as model organisms to demonstrate the method. A unified protein preparation protocol was developed for both cell types to streamline the benchtop process and aid future automation. Cells and spores were lysed and proteins solubilized using a combination of thermal and chemical lysis methods. Reducing agents, necessary to solubilize spore proteins, were eliminated using a small-scale rapid size-exclusion chromatography step to eliminate interference with down-stream protein labeling. This approach was found to be compatible with nonspore cells (i.e., vegetative cells) as well, not adversely impacting the protein signatures. Data are presented demonstrating distinct CGE protein signatures for our model organisms, suggesting the potential for discrimination of organisms on the basis of empirical protein patterns. The goal of this work is to develop a fast and field-portable method for characterizing bacteria via their proteomes. PMID:18008300

  14. Self-organized criticality in proteins: Hydropathic roughening profiles of G-protein-coupled receptors

    Phillips, J. C.

    2013-03-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein conformational functionality is often dominated by long-range hydrophobic or hydrophilic interactions which both drive protein compaction and mediate protein-protein interactions. Superfamily transmembrane G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome; their amino acid sequences form the largest database for protein-membrane interactions. While there are now structural data on the heptad transmembrane structures of representatives of several heptad families, here we show how fresh insights into global and some local chemical trends in GPCR properties can be obtained accurately from sequences alone, especially by algebraically separating the extracellular and cytoplasmic loops from transmembrane segments. The global mediation of long-range water-protein interactions occurs in conjunction with modulation of these interactions by roughened interfaces. Hydropathic roughening profiles are defined here solely in terms of amino acid sequences, and knowledge of protein coordinates is not required. Roughening profiles both for GPCR and some simpler protein families display accurate and transparent connections to protein functionality, and identify natural length scales for protein functionality.

  15. [SHIFTS IN URINE PROTEIN PROFILE DURING DRY IMMERSION].

    Pastushkova L Kh; Kononikhin, A S; Tiys, E S; Nosovsky, A M; Dobrokhotov, I V; Ivanisenko, V A; Nikolaev, E N; Novoselova, N M; Custaud, M A; Larina, I M

    2015-01-01

    The study was aimed at tracking the proteomic profile of urine in 8 normal volunteers to 5-day dry immersion (DI). The proteome composition was determined by chromatography-mass spectrometry on high-efficient on-line liquid nano chromatograph Agilent 1100; complementary information about the protein spectra was obtained by dint of mass-spectrometer MaXis Impact 4G and hybrid mass-spectrometer LTQ-FT. Functional associations between proteins and biological functions were analyzed using computer system ANDCell (Associative Networks Discovery in Cells). A total of 256 proteins were identified; for 43 proteins difference in the detection rate during the baseline data collection and on DI day 4 exceeded 20%. PMID:26554129

  16. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer

  17. Protein profiles distinguish stable and progressive chronic lymphocytic leukemia.

    Huang, Pauline Y; Mactier, Swetlana; Armacki, Natalie; Giles Best, O; Belov, Larissa; Kaufman, Kimberley L; Pascovici, Dana; Mulligan, Stephen P; Christopherson, Richard I

    2016-05-01

    Patients with a stable chronic lymphocytic leukemia (CLL) double their blood lymphocyte count in >5 years, but may develop progressive disease with lymphocytes doubling in selected reaction monitoring) using extracts of purified CD19(+) CLL cells from patients (n = 50). Hierarchical clustering of these protein profiles showed two clusters of patients that correlated with progressive and stable CLL, providing signatures that should be useful for triaging patients. Some of the proteins in the progressive cluster have not been linked with CLL, for example, glutamate dehydrogenase 1 and transcription intermediary factor 1-beta. PMID:26422656

  18. Phylogeny reconstruction based on protein phylogenetic profiles of organisms

    2003-01-01

    With the coming of the Post Genomic Era, more and more genomes have been sequenced and it has become possible to study phylogeny reconstruction at genome level. The concept of protein phylogenetic profiles of organisms is defined in this work which is used in phylogeny reconstruction by proteome comparisons. This method is more stable than the prevailing molecular systematics methods and can be used widely. It will develop very fast with the rapid progress in genome sequencing.

  19. Protein Expression Profiling in the Spectrum of Renal Cell Carcinomas

    Vladimir A Valera, Elsa Li-Ning-T, Beatriz A Walter, David D. Roberts, W M Linehan, Maria J Merino

    2010-01-01

    Full Text Available In this study, we aimed to evaluate the protein expression profile of a spectrum of renal cell carcinomas (RCC to find potential biomarkers for disease onset and progression and therefore, prospective therapeutic targets. A 2D-gel based proteomic analysis was used to outline differences in protein levels among different subtypes of renal cell carcinomas, including clear cell carcinomas, papillary lesions, chromophobe tumors and renal oncocytomas. Spot pattern was compared to the corresponding normal kidney from the same patients and distinctive, differentially expressed proteins were characterized by mass spectrometry. Twenty-one protein spots were found differentially expressed between clear cell RCC and normal tissue and 38 spots were found expressed in chromophobe tumors. Eleven proteins were identified, with most differentially expressed -by fold change- between clear cell tumors and the corresponding normal tissue. Two of the identified proteins, Triosephosphate isomerase 1 (TPI-1 and Heat Shock protein 27 (Hsp27, were further validated in a separate set of tumors by immunohistochemistry and expression levels were correlated with clinicopathologic features of the patients. Hsp27 was highly expressed in 82% of the tumors used for validation, and all cases showed strong immunoreactivity for TPI-1. In both Hsp27 and TPI-1, protein expression positively correlated with histologic features of the disease. Our results suggest that the subjacent cytogenetic abnormalities seen in different histological types of RCC are followed by specific changes in protein expression. From these changes, Hsp27 and TPI-1 emerged as potential candidates for the differentiation and prognosis in RCC.

  20. Fold homology detection using sequence fragment composition profiles of proteins.

    Solis, Armando D; Rackovsky, Shalom R

    2010-10-01

    The effectiveness of sequence alignment in detecting structural homology among protein sequences decreases markedly when pairwise sequence identity is low (the so-called "twilight zone" problem of sequence alignment). Alternative sequence comparison strategies able to detect structural kinship among highly divergent sequences are necessary to address this need. Among them are alignment-free methods, which use global sequence properties (such as amino acid composition) to identify structural homology in a rapid and straightforward way. We explore the viability of using tetramer sequence fragment composition profiles in finding structural relationships that lie undetected by traditional alignment. We establish a strategy to recast any given protein sequence into a tetramer sequence fragment composition profile, using a series of amino acid clustering steps that have been optimized for mutual information. Our method has the effect of compressing the set of 160,000 unique tetramers (if using the 20-letter amino acid alphabet) into a more tractable number of reduced tetramers (approximately 15-30), so that a meaningful tetramer composition profile can be constructed. We test remote homology detection at the topology and fold superfamily levels using a comprehensive set of fold homologs, culled from the CATH database that share low pairwise sequence similarity. Using the receiver-operating characteristic measure, we demonstrate potentially significant improvement in using information-optimized reduced tetramer composition, over methods relying only on the raw amino acid composition or on traditional sequence alignment, in homology detection at or below the "twilight zone". PMID:20635424

  1. Proteomic dissection of biological pathways/processes through profiling protein-protein interaction networks

    2010-01-01

    Cellular functions, either under the normal or pathological conditions or under different stresses, are the results of the coordinated action of multiple proteins interacting in macromolecular complexes or assemblies. The precise determination of the specific composition of protein complexes, especially using scalable and high-throughput methods, represents a systematic approach toward revealing particular cellular biological functions. In this regard, the direct profiling protein-protein interactions (PPIs) represent an efficient way to dissect functional pathways for revealing novel protein functions. In this review, we illustrate the technological evolution for the large-scale and precise identification of PPIs toward higher physiologically relevant accuracy. These techniques aim at improving the efficiency of complex pull-down, the signal specificity and accuracy in distinguishing specific PPIs, and the accuracy of identifying physiological relevant PPIs. A newly developed streamline proteomic approach for mapping the binary relationship of PPIs in a protein complex is introduced.

  2. Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

    Rundle William T

    2008-06-01

    Full Text Available Abstract Background Penicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase, but at body temperature (37°C, a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei. Results Whole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein

  3. Protein content and amino acids profile of pseudocereals.

    Mota, Carla; Santos, Mariana; Mauro, Raul; Samman, Norma; Matos, Ana Sofia; Torres, Duarte; Castanheira, Isabel

    2016-02-15

    Quinoa (Chenopodium quinoa), amaranth (Amaranthus caudatus) and buckwheat (Fagopyrum esculentum) represent the main protein source in several diets, although these pseudocereals are not currently present in the FCDB nutrient profile information. The aim of this work is to characterise the AA profile of these pseudocereals and compare them with rice. Total protein content revealed to vary from 16.3g/100g (quinoa Salta) to 13.1g/100g (buckwheat) and lower values were found in rice samples (6.7g/100g). For pseudocereals the most abundant essential AA was leucine. Quinoa-Salta evidences the highest leucine content (1013mg/100g) and the minor methionine content (199mg/100g). Buckwheat was the cereal with the highest phenylalanine content (862mg/100g). Rice (Oryza sativa) presents the lowest content for all AA. Results showed pseudocereals as the best source of AA. EuroFIR guidelines where strictly followed and proved to be a crucial tool to guarantee data interchangeability and comparability. PMID:26433287

  4. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    Guang-Jian Jiang

    Full Text Available It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  5. Design and synthesis of ATP-based nucleotide analogues and profiling of nucleotide-binding proteins

    Wolters, Justina. C.; Roelfes, Johannes; Poolman, Bert

    2011-01-01

    Two nucleotide-based probes were designed and synthesized in order to enrich samples for specific classes of proteins by affinity-based protein profiling. We focused on the profiling of adenine nucleotide-binding proteins. Two properties were considered in the design of the probes: the bait needs to

  6. Automatic selection of reference taxa for protein-protein interaction prediction with phylogenetic profiling

    Simonsen, Martin; Maetschke, S.R.; Ragan, M.A.

    2012-01-01

    Motivation: Phylogenetic profiling methods can achieve good accuracy in predicting protein–protein interactions, especially in prokaryotes. Recent studies have shown that the choice of reference taxa (RT) is critical for accurate prediction, but with more than 2500 fully sequenced taxa publicly......: We present three novel methods for automating the selection of RT, using machine learning based on known protein–protein interaction networks. One of these methods in particular, Tree-Based Search, yields greatly improved prediction accuracies. We further show that different methods for constituting...

  7. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    Villamor, J. G.; Kaschani, F.; Colby, T; Oeljeklaus, J.; Zhao, D; Kaiser, M.; Patricelli, M. P.; R. A. L. van der Hoorn

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding prot...

  8. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  9. Analysis of Proximate and Protein Profile of Kefir from Fermented Goat and Cow Milk

    Erwin Hidayat; Irna Kinayungan W; Muhammad Irhas; Fathurrahman Sidiq

    2015-01-01

    This research aims to analyze the characteristics of proximate and protein profile in kefir from fermented goat milk and cow milk with different concentration of kefir grains. The research design was true experimental with Completely Randomized Design (CRD) of 3 repetitions. The research procedures consisted of kefir production, proximate analysis and protein profile characterization. Proximate assay result was analyzed by using LSD, whereas the protein profile was analyzed by descriptive qua...

  10. Strain-dependent profile of misfolded prion protein aggregates.

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration. PMID:26877167

  11. Study of protein and metabolic profile of sugarcane workers

    Full text: The National Alcohol Program (Proalcool) is a successful Brazilian renewable fuel initiative aiming to reduce the country's oil dependence. Producing ethanol from sugar cane, the program has shown positive results although accompanied by potential damage. The environmental impact mainly derives from the particulate matter emissions due to sugarcane burning, which is potentially harmful to human health. The physical activity of sugarcane workers is repetitive and exhaustive and is carried out in presence of dust, smoke and soot. The efforts by the sugarcane workers during the labor process result in increased risks of nervous, respiratory and cardiovascular system diseases and also in premature death. The aim of the present study was to investigate the effect of occupational stress on protein and metabolic profile of sugarcane workers. Forty serum samples were analyzed by 1-DE and LC MS/MS proteomic shotgun strategy and nuclear magnetic resonance (NMR). A set of proteins was found to be altered in workers after crops when compared with controls. The analysis of NMR spectra by Chenomx also showed differences in the expression of metabolites. For example, lactate displayed higher levels in control subjects than in sugarcane workers, and vice versa for the acetate. The concentrations of the two metabolites were lower after the crop, except in the case of acetate, which remained uniform in the control subjects before and after the crop. The present findings can have important application for rational designs of preventive measures and early disease detection in sugarcane workers. (author)

  12. Study of protein and metabolic profile of sugarcane workers

    Polachini, G.M.; Tajara, E.H. [Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP), SP (Brazil); Santos, U.P. [Universidade de Sao Paulo (USP), SP (Brazil); Zeri, A.C.M.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil)

    2012-07-01

    Full text: The National Alcohol Program (Proalcool) is a successful Brazilian renewable fuel initiative aiming to reduce the country's oil dependence. Producing ethanol from sugar cane, the program has shown positive results although accompanied by potential damage. The environmental impact mainly derives from the particulate matter emissions due to sugarcane burning, which is potentially harmful to human health. The physical activity of sugarcane workers is repetitive and exhaustive and is carried out in presence of dust, smoke and soot. The efforts by the sugarcane workers during the labor process result in increased risks of nervous, respiratory and cardiovascular system diseases and also in premature death. The aim of the present study was to investigate the effect of occupational stress on protein and metabolic profile of sugarcane workers. Forty serum samples were analyzed by 1-DE and LC MS/MS proteomic shotgun strategy and nuclear magnetic resonance (NMR). A set of proteins was found to be altered in workers after crops when compared with controls. The analysis of NMR spectra by Chenomx also showed differences in the expression of metabolites. For example, lactate displayed higher levels in control subjects than in sugarcane workers, and vice versa for the acetate. The concentrations of the two metabolites were lower after the crop, except in the case of acetate, which remained uniform in the control subjects before and after the crop. The present findings can have important application for rational designs of preventive measures and early disease detection in sugarcane workers. (author)

  13. Ribo-Proteomics Approach to Profile RNA-Protein and Protein-Protein Interaction Networks.

    Yeh, Hsin-Sung; Chang, Jae-Woong; Yong, Jeongsik

    2016-01-01

    Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses. PMID:26965265

  14. FRAKSIONASI DAN PENENTUAN PROFIL PROTEIN BUNGKIL KELAPA DENGAN SDS-PAGE [Fractionation and Profiling of Copra Meal’s Protein by Using SDS-PAGE

    Agus Danang Wibowo1); Suhartono, Maggy T; Patuan L. P. Siagian2)

    2012-01-01

    The aim of this research was to extract proteins from the copra meal, base on their solubility and analysis of the protein profiles by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) This research was conducted in two stages, protein fractionation and molecular weight estimation using SDS PAGE. The fractionation was conducted by non-enzymatic treatment and enzymatic (mannanase) treatment. Kjeldahl analysis showed that the protein content of copra meal was 18.52% of dry b...

  15. Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins

    Cheng Chung-Hsien

    2010-08-01

    Full Text Available Abstract Background Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase, both fused to glutathione S-transferase (GST and polyionic peptide (5D or 5R. Results Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA. Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Conclusions Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the

  16. Serum Protein Profile Study of Clinical Samples Using High Performance Liquid Chromatography-Laser Induced Fluorescence

    Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya;

    2009-01-01

    The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for...

  17. Protein Profile study of clinical samples using Laser Induced Fluorescence as the detection method

    Karemore, Gopal Raghunath; Raja, Sujatha N.; Rai, Lavanya;

    2009-01-01

      Protein profiles of tissue homogenates were recorded using HPLC separation and LIF detection method. The samples were collected from volunteers with clinically normal or cervical cancer conditions. It is shown that the protein profile can be classified as belonging to malignant or normal state ...

  18. FRAKSIONASI DAN PENENTUAN PROFIL PROTEIN BUNGKIL KELAPA DENGAN SDS-PAGE [Fractionation and Profiling of Copra Meal’s Protein by Using SDS-PAGE

    Agus Danang Wibowo1

    2012-06-01

    Full Text Available The aim of this research was to extract proteins from the copra meal, base on their solubility and analysis of the protein profiles by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE This research was conducted in two stages, protein fractionation and molecular weight estimation using SDS PAGE. The fractionation was conducted by non-enzymatic treatment and enzymatic (mannanase treatment. Kjeldahl analysis showed that the protein content of copra meal was 18.52% of dry basis. Protein fractionation could separate the copra meal’s proteins based on their solubility. Albumin was solubilized by water, globulin by NaCl 5%, glutelin by NaOH 0.02M, and prolamin by ethanol 70%. Fractionation using non-enzymatic treatment resulted in 64% of albumin, 39.25% of globulin, 15.27% of glutelin, and 38.84% of prolamin fractions. On the other hand, fractionation using enzymatic treatment resulted different protein profile, giving 28.17%, 39.44%, 10.91%, and 21.48% of the respective proteins. Characterization using SDS PAGE showed that the protein profile from the non-enzymatic was different from that of the enzymatic treatment. In conclusion, we found that the proteins extracted from the copra meal with and without mannanase showed variation in the range of molecular weights: 45.1-66,0 kDa and 66.1-97.4 kDa, respectively.

  19. DNA-Mediated Detection and Profiling of Protein Complexes

    Hammond, Maria

    2013-01-01

    Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding...

  20. Protein profile study of the cervical cancer using HPLC-LIF

    Sujatha; Rai, Lavanya; Krishnanand, B. R.; Mahato, K. K.; Kartha, V. B.; C, Santhosh

    2006-02-01

    Optical methods and proteomics investigations are becoming promising approaches for early detection of many diseases, which remain clinically silent for long periods. We have used efficient High Performance Liquid Chromatography (HPLC) separation combined with highly sensitive laser induced fluorescence detection of proteins present in clinical samples for diagnostic applications in cervical cancer. The protein profile and the fluorescence of individual proteins were simultaneously recorded using our HPLC-LIF system. Protein profiles (Chromatogram) of serum from normal male and female volunteers with and without tobacco habits, and malignant serum samples were studied. Protein profiles were also recorded for lysates of exfoliated cells collected from Pap smear of normal and cancer patients. The protein profile patterns were subjected to Principal component Analysis. Discrimination of normal and malignant samples were achieved with very high sensitivity and specificity.

  1. Changes of protein profile during the brewing process

    Benkovská, D. (Dagmar); Flodrová, D. (Dana); Bobálová, J. (Janette)

    2012-01-01

    Our work was focused on the protein identification in individual stages of brewing process. The greatest attention was paid to the proteins that resist the harsh conditions applied during brewing and therefore may influence various beer properties. These proteins (nsLTPs, protein Z and group of protease/alpha-amylase inhibitors) belong to the group of PRs.

  2. Modification of resolution in capillary electrophoresis for protein profiling in identification of genetic modification in foods

    Latoszek, A.; Cifuentes, Alejandro

    2011-01-01

    The capillary electrophoresis with UV detection was employed for protein profiling in extracts from maize and soybeans. Modifications of back-ground electrolyte and coating the capillary wall with polybrene was employed in order to decrease the protein adsorption on the capillary walls. The obtained protein profiles were compared for transgenic and non-transgenic variants, showing in some cases significant changes that might be employed for identification of genetic modifications ...

  3. Reproducibility of mass spectrometry based protein profiles for diagnosis of breast cancer across clinical studies

    Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E; Cold, Søren; Mogensen, Ole; Kruse, Torben; Jensen, Ole Nørregaard; Madsen, Jonna S

    2008-01-01

    Serum protein profiling by mass spectrometry has achieved attention as a promising technology in oncoproteomics. We performed a systematic review of published reports on protein profiling as a diagnostic tool for breast cancer. The MEDLINE, EMBASE, and COCHRANE databases were searched for origina...... indicating some convergence toward a set of common discriminating, reproducible peaks for breast cancer. These peaks should be further characterized for identification of the protein identity and validated as biomarkers for breast cancer....

  4. Protein abundance profiling of the Escherichia coli cytosol

    Ishihama, Y.; Schmidt, T.; Rappsilber, J.;

    2008-01-01

    sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed em...... between protein and mRNA abundance in E. coli cells. Conclusion: Abundance measurements for more than 1000 E. coli proteins presented in this work represent the most complete study of protein abundance in a bacterial cell so far. We show significant associations between the abundance of a protein and its...

  5. Differences in tomato seed protein profiles obtained by SDS-PAGE analysis

    Miskoska-Milevska Elizabeta; Dimitrievska Blagica; Poru Koo; Popovski Zoran T.

    2008-01-01

    The protein profiles of tomato seeds from sub-species ( subsp. cultum Brezh., subsp. subspontaneum Brezh. and subsp. spontaneum Brezh.) were analyzed using SDS-PAGE technique. Electrophoreograms and denzitograms of total, soluble and non-soluble proteins of 31 different samples have showed quantitative and qualitative differences. Qualitative differences in electrophoregrams of total seed proteins refer to protein fragments in zone A (114 kDa, 83 kDa and 65 kDa) and protein fragment in zone C...

  6. Label-free detection of proteins in ternary mixtures using surface-enhanced Raman scattering and protein melting profiles

    Keskin, Sercan; Efeoğlu, Esen; Keçeci, Kaan; Çulha, Mustafa

    2013-03-01

    The multiplex detection of biologically important molecules such as proteins in complex mixtures has critical importance not only in disease diagnosis but also in other fields such as proteomics and biotechnology. Surface-enhanced Raman scattering (SERS) is a powerful technique for multiplex identification of molecular components in a mixture. We combined the multiplexing power of SERS and heat denaturation of proteins to identify proteins in ternary protein mixtures. The heat denaturation profiles of four model blood proteins, transferrin, human serum albumin, fibrinogen, and hemoglobin, were studied with SERS. Then, two ternary mixtures of these four proteins were used to test the feasibility of the approach. It was demonstrated that unique denaturation profiles of each protein could be used for their identification in the mixture.

  7. Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

    Juncker, Agnieszka; Larsen, Mette Voldby; Weinhold, Nils; Nielsen, Morten; Brunak, Søren; Lund, Ole

    2009-01-01

    -scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly...

  8. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  9. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  10. Protein abundance profiling of the Escherichia coli cytosol

    Mann Matthias

    2008-02-01

    Full Text Available Abstract Background Knowledge about the abundance of molecular components is an important prerequisite for building quantitative predictive models of cellular behavior. Proteins are central components of these models, since they carry out most of the fundamental processes in the cell. Thus far, protein concentrations have been difficult to measure on a large scale, but proteomic technologies have now advanced to a stage where this information becomes readily accessible. Results Here, we describe an experimental scheme to maximize the coverage of proteins identified by mass spectrometry of a complex biological sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal proteins. Proteins involved in energy metabolism as well as those with binding function were also found in high copy number while proteins annotated with the terms metabolism, transcription, transport, and cellular organization were rare. The barrel-sandwich fold was found to be the structural fold with the highest abundance. Highly abundant proteins are predicted to be less prone to aggregation based on their length, pI values, and occurrence patterns of hydrophobic stretches. We also find that abundant proteins tend to be predominantly essential. Additionally we observe a significant correlation between protein and mRNA abundance in E. coli cells. Conclusion Abundance measurements for more than 1000 E. coli proteins presented in this work

  11. Analysis of Proximate and Protein Profile of Kefir from Fermented Goat and Cow Milk

    Erwin Hidayat

    2015-09-01

    Full Text Available This research aims to analyze the characteristics of proximate and protein profile in kefir from fermented goat milk and cow milk with different concentration of kefir grains. The research design was true experimental with Completely Randomized Design (CRD of 3 repetitions. The research procedures consisted of kefir production, proximate analysis and protein profile characterization. Proximate assay result was analyzed by using LSD, whereas the protein profile was analyzed by descriptive qualitative method. Based on the analysis of kefir proximate levels, the kefir grain (5% showed the highest proximate level of both kefirs from goat milk and cow milk. The analysis of protein profile of cow milk kefir showed 75 kDa of protein ribbon, while the goat milk kefir showed 48 kDa, 60 kDa and 75 kDa. Therefore it can be concluded that the proximate level of goat and cow milk kefir with different concentration of kefir grains showed significant differences in the nutrition content as well as its protein profiles.Tujuan dari penelitian ini adalah menganalisis karakteristik proksimat dan profil protein pada kefir hasil fermentasi susu kambing dan susu sapi dengan konsentrasi biji kefir yang berbeda-beda. Penelitian ini adalah eksperimen murni, dengan Rancangan Acak Lengkap (RAL 3 kali ulangan. Prosedur penelitian meliputi pembuatan kefir, analisis proksimat dan profil protein. Data hasil proksimat dianalisi uji BNT, sedangkan profil protein dianalisis deskriptif kualitatif. Berdasarkan analisis kadar proksimat kefir, kefir grains 5% menunjukan kadar proksimat paling tinggi baik pada kefir susu kambing dan susu sapi. Sedangkan analisis profil protein kefir susu sapi menunjukan pita protein 75 kDa, pada kefir susu kambing yaitu 48 kDa, 60 kDa dan 75 kDa. Simpulan dari penelitian ini bahwa kadar proksimat kefir susu kambing dan susu sapi dengan konsentrasi kefir grains yang berbeda menunjukan perbedaan kandungan yang berbeda secara signifikan dengan

  12. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

    Williams, Christopher C.; Jan, Calvin H.; Weissman, Jonathan S.

    2014-01-01

    Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of protein...

  13. Buffalo milk: proteins electrophoretic profile and somatic cell count

    S. Mattii; B. Tommei; Pasquini, M.

    2011-01-01

    Water buffalo milk differs from the cow’s milk for greater fat and protein content, very important features in cheese making. Proteins, casein and whey-proteins in particular, are the most important factors determining cheese yield. Several previous research discussed the rule of SCC in cow milk production (Varisco, 1999) and the close relationship existing between cow’s milk cheese yield and somatic cell count (Barbano, 2000). In particular the inverse correlation between cheese ...

  14. Proteomics of protein expression profiling in tissues with different radiosensitivity

    The purpose of this study was to identify Radiosensitivity of proteins in tissues with different radiosensitivity. C3H/HeJ mice were exposed to 10 Gy. The mice were sacrifiud 8 hrs after radiation. Their spleen and liver tissues were collected and analyzed histologically for apoptosis. The expressions of radiosusceptibility protein were analyzed by 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The peak of apoptosis levels were 35.3 ± 1.7% in spleen and 0.6 ± 0.2% in liver at 8 hrs after radiation. Liver, radioresistant tissues, showed that the levels of ROS metabolism related to proteins such as cytochromm c, glutathione S transferase, NADH dehydrogenase, riken cDNA and peroxiredoxin VI increased after radiation. The expression of cytochrome c increased significantly in spleen and liver tissues after radiation. In spleen, radiosensitivity tissue, the identified proteins showed a significantly quantitative alteration following radiation. It was categorized as signal transduction, apoptosis, cytokine, Ca signal related protein, stress-related protein, cytoskeletal regulation, ROS metabolism, and others. Differences of radiation-induced apoptosis by tissues specifted were coupled with the induction of related radiosensitivity and radioresistant proteins. The result suggests that apoptosis relate protein and redox proteins play important roles in this radiosusceptibility

  15. Whole-Cell Protein Profiles of Disintegrated Freshwater Green Algae and Cyanobacterium

    Samek, Dušan; Mišurcová, Ladislava; Machů, Ludmila; Buňková, Leona; Minařík, Antonín; Fišera, Miroslav

    2016-01-01

    The influence of cultivation methods and postharvesting treatment on protein profiles of green freshwater microalgae Chlorella kessleri, Scenedesmus quadricauda, and Chlorella sp. and cyanobacterium Spirulina platensis were evaluated. The comparison of protein profiles in algal biomass originated from the autotrophic cultivation in an outdoor open circulating cascade-type cultivation apparatus in thin-layer, a solar photobioreactor, and from the heterotrophic cultivation regime in a fermenter...

  16. Reproducibility of mass spectrometry based protein profiles for diagnosis of ovarian cancer across clinical studies

    Øgendahl Callesen, Anne Kjærgaard; Mogensen, Ole; Jensen, Andreas K; Kruse, Torben A; Martinussen, Torben; Jensen, Ole N; Madsen, Jonna S

    2012-01-01

    The focus of this systematic review is to give an overview of the current status of clinical protein profiling studies using MALDI and SELDI MS platforms in the search for ovarian cancer biomarkers. A total of 34 profiling studies were qualified for inclusion in the review. Comparative analysis o...... article is part of a Special Issue entitled: Clinical Proteomics SI: Clinical Proteomics....

  17. Comparative protein profiles of Butea superba tubers under seasonal changes.

    Leelahawong, Chonchanok; Srisomsap, Chantragan; Cherdshewasart, Wichai; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Sangvanich, Polkit

    2016-07-01

    Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future. PMID:27198528

  18. Hierarchical partitioning of metazoan protein conservation profiles provides new functional insights.

    Jonathan Witztum

    Full Text Available The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles. We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO analysis tool, we explore functional enrichment of the "universal proteins", those with homologues in all 17 other species, and of the "non-universal proteins". A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the "tree of life" (TOL consistent, as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the "life style" of the related clades. Most previous approaches for studying function and conservation are "bottom up", studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is "top down". We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life.

  19. Hierarchical partitioning of metazoan protein conservation profiles provides new functional insights.

    Witztum, Jonathan; Persi, Erez; Horn, David; Pasmanik-Chor, Metsada; Chor, Benny

    2014-01-01

    The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles). We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO) analysis tool, we explore functional enrichment of the "universal proteins", those with homologues in all 17 other species, and of the "non-universal proteins". A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the "tree of life" (TOL consistent), as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the "life style" of the related clades. Most previous approaches for studying function and conservation are "bottom up", studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is "top down". We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life. PMID:24594619

  20. Serum protein profiles as potential biomarkers for infectious disease status in pigs

    Koene Miriam GJ

    2012-03-01

    Full Text Available Abstract Background In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circovirus-associated disease (PCVAD, consisting of Porcine Circovirus type 2 (PCV2 infection in combination with either Porcine Parvovirus (PPV or Porcine Reproductive and Respiratory Syndrome virus (PRRSV. Sera were collected before and after onset of clinical signs at day 0, 5 and 19 post infection. Serum protein profiles were evaluated against sera from non-infected control animals. Results Protein profiles were generated by SELDI-TOF mass spectrometry in combination with the Proteominer™ technology to enrich for low-abundance proteins. Based on these protein profiles, the experimentally infected pigs could be classified according to their infectious disease status. Before the onset of clinical signs 88% of the infected animals could be classified correctly, after the onset of clinical sigs 93%. The sensitivity of the classification appeared to be high. The protein profiles could distinguish between separate infection models, although specificity was moderate to low. Classification of PCV2/PRRSV infected animals was superior compared to PCV2/PPV infected animals. Limiting the number of proteins in the profiles (ranging from 568 to 10 had only minor effects on the classification performance. Conclusions This study shows that serum protein profiles have potential for detection and identification of viral infections in pigs before clinical signs of the disease become visible.

  1. Correlations between RNA and protein expression profiles in 23 human cell lines

    Pontén Fredrik

    2009-08-01

    Full Text Available Abstract Background The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays and protein profiles (determined immunohistochemically in tissue microarrays of 1066 gene products in 23 human cell lines. Results A high mean correlation coefficient (0.52 was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

  2. Protein and lipid MALDI profiles classify breast cancers according to the intrinsic subtype

    Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of common solid tumors. Using recently developed MALDI matrices for lipid profiling, we evaluated whether direct tissue MALDI MS analysis on proteins and lipids may classify human breast cancer samples according to the intrinsic subtype. Thirty-four pairs of frozen, resected breast cancer and adjacent normal tissue samples were analyzed using histology-directed, MALDI MS analysis. Sinapinic acid and 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument. Protein and lipid profiles distinguish cancer from adjacent normal tissue samples with the median prediction accuracy of 94.1%. Luminal, HER2+, and triple-negative tumors demonstrated different protein and lipid profiles, as evidenced by permutation P values less than 0.01 for 0.632+ bootstrap cross-validated misclassification rates with all classifiers tested. Discriminatory proteins and lipids were useful for classifying tumors according to the intrinsic subtype with median prediction accuracies of 80.0-81.3% in random test sets. Protein and lipid profiles accurately distinguish tumor from adjacent normal tissue and classify breast cancers according to the intrinsic subtype

  3. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage.

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-15

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1×, DE3×, ME3×, NEL3×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P=0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P=0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P=0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P=0.09), RD of PB2 (29 vs. 62 g/kg CP, P=0.02), RD of CB2 (251 vs. 198 g/kg DM, P=0.06), RD of CB3 (236 vs. 261 g/kg CHO, P=0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P=0.06), and metabolic bioenergy (P=0.095). As to protein molecular structure, there were differences in protein structure 1st and 2nd amide groups (P0

  4. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-01

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

  5. Multidimensional profiling of cell surface proteins and nuclear markers

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  6. Optimization of translation profiles enhances protein expression and solubility.

    Anne-Katrin Hess

    Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  7. Differences in tomato seed protein profiles obtained by SDS-PAGE analysis

    Miskoska-Milevska Elizabeta

    2008-01-01

    Full Text Available The protein profiles of tomato seeds from sub-species ( subsp. cultum Brezh., subsp. subspontaneum Brezh. and subsp. spontaneum Brezh. were analyzed using SDS-PAGE technique. Electrophoreograms and denzitograms of total, soluble and non-soluble proteins of 31 different samples have showed quantitative and qualitative differences. Qualitative differences in electrophoregrams of total seed proteins refer to protein fragments in zone A (114 kDa, 83 kDa and 65 kDa and protein fragment in zone C (17 kDa. Qualitative differences in electrophoregrams of soluble seed proteins refer to protein fragment in zone A (94 kDa. Qualitative differences in electrophoregrams of nonsoluble seed proteins refer to protein fragments with molecular weights of: 210 kDa, 85 kDa, 67 kDa and 26 kDa.

  8. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    Kaake, Robyn M; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand ho...

  9. Buffalo milk: proteins electrophoretic profile and somatic cell count

    S. Mattii

    2011-03-01

    Full Text Available Water buffalo milk differs from the cow’s milk for greater fat and protein content, very important features in cheese making. Proteins, casein and whey-proteins in particular, are the most important factors determining cheese yield. Several previous research discussed the rule of SCC in cow milk production (Varisco, 1999 and the close relationship existing between cow’s milk cheese yield and somatic cell count (Barbano, 2000. In particular the inverse correlation between cheese yields and somatic cells’content have been demonstrated. In Italy the regulation in force DPR 54/97 acknowledges what expressed in EEC 46/92 Directive (Tripodi, 1999 without fixing the limit threshold of somatic cells for buffalo’s milk....

  10. Determination of heat capacity of unfolding for marginally stable proteins from a single temperature induced protein unfolding profile

    A reliable estimation of heat capacity of denaturation (ΔCp) is necessary to calculate the free energy of unfolding of proteins. For marginally stable proteins, such as mutants of a protein or proteins at low pH or under denaturating conditions, the pre-transition region is not fully populated by the native state. Analysis of differential scanning calorimeter (DSC) data under such conditions may not yield a reliable value of ΔCp and other associated thermodynamic parameters of unfolding. Analysis of denaturation profiles of (a) cytochrome c at pH 2.5, 3 and 8 and (b) myoglobin at pH 4, show that an accurate value of ΔCp can be extracted from a single unfolding profile obtained spectroscopically by including low temperature data.

  11. Changes of Electrophoretic Protein Profiles of Smoked and Marinated Rainbow Trout (Oncorhynchus mykiss) During Refrigerated Storage

    BAYLAN, Makbule; MAZI, Gamze; ÖZCAN, Numan; ÖZCAN, Bahri Devrim; AKAR, Mustafa; Coşkun, Ali

    2015-01-01

    In this study, we aimed to determine the changes of electrophoretic protein profiles of smoked and marinated rainbow trout (Oncorhynchus mykiss) during refrigerated storage. Changes in muscle proteins during 9 weeks refrigerated storage of raw, smoked and marinated trout samples have been examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and densitometric analysis revealed that intensity and the number of some protein bands were reduced while the b...

  12. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  13. Profile of total protein, albumin, globulin and albumin globulin ratio in bulls

    Ida Zahidah Irfan; Esfandiari, A; C Choliq

    2014-01-01

    Determination of serum total protein concentration and main fractions (albumin and globulin) can be used as an important diagnostic tool in clinical biochemistry. Several factors can affect the concentration of total protein, albumin, globulin and albumin globulin ratio (A/G). The aim of this study is to obtain serum protein profiles, albumin, globulin and A/G ratio based on breed, age and BCS (body condition score). Blood samples from 160 bulls were collected. Blood chemistry were analyzed ...

  14. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  15. Study of protein profile in individual stages of brewing process

    Benkovská, Dagmar; Flodrová, Dana; Psota, V.; Bobálová, Janette

    Berlin : Freie Universität Berlin, 2011. s. 350. [Proteomic Forum 2011. 03.04.2011-07.04.2011, Berlin] R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : proteins * barley * brewing Subject RIV: CB - Analytical Chemistry, Separation

  16. Milk protein profiles in response to Streptococcus agalactiae subclinical mastitis in dairy cows.

    Pongthaisong, Pongphol; Katawatin, Suporn; Thamrongyoswittayakul, Chaiyapas; Roytrakul, Sittiruk

    2016-01-01

    The objective of this study was to investigate the milk protein profiles of normal milk and those of milk during the course of subclinical mastitis, caused by natural Streptococcus agalactiae infection. Two-dimensional gel electrophoresis and liquid chromatography mass spectrometry were used to assess protein profiles and to identify the proteins. The results showed that S. agalactiae subclinical mastitis altered the protein profiles of milk. Following Mascot database matching, 11 and 12 protein types were identified in the milk collected from healthy and S. agalactiae subclinical mastitic udders, respectively. The distinct presence of the antibacterial protein cathelicidin-1 was detected in infected milk samples, which in turn was highly correlated to the severity of subclinical mastitis as represented by the milk somatic cell count (r = 0.616), but not the bacterial count. The protein profile of milk reveals changes in the host response to S. agalactiae intramammary infection; cathelicidin-1 could therefore serve as a biomarker for the detection of subclinical mastitis in dairy cows. PMID:26632331

  17. Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

    Yi Feng; Zhong-Min Tian; Ming-Xi Wan; Zhao-Bin Zheng

    2007-01-01

    AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.METHODS: Total proteins from human hepatocarcinomacell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite.Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)and database searching.RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin,endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein Ⅰ,peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

  18. Protein profile of Chlamydophila abortus isolates from Kerala, India

    Binu K Mani

    Full Text Available Chlamydiae are of microbiological interest because of their mode of interaction with eukaryotic host cells and their specialized life cycle with unique features of parasitism. Reports regarding prevalence of infections of Chlamydophila abortus, the causative organism for chlamydial abortions in livestock, was the basis of the study. Two isolates, one each from cattle and goat abortion along with a reference isolate, were used for characterization with Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE. Elementary bodies infected Mc Coy cells, harvested from bottle cultures were disrupted by Teflon coated magnetic pellet. Urografin-76 diluted with Tris-Potassium hydrochloride was used for purification of Elementary bodies of Chlamydophila abortus organism. On protein estimation of Elementary bodies by Biuret method, all the three isolates revealed protein concentration between 500-1000 mg/100ml, which were sufficient for electrophoresis. Ten percent of resolving gel and five percent of stacking gel of polyacrylamide in which 10g of processed isolate samples along with standard protein marker and Mc Coy cell protein (control were electrophoresed. Using Alpha Imager Gel Documentation System, the protein bands were analyzed. Twelve bands each for local bovine isolate and reference isolate were noticed while only 10 bands were there in the caprine isolate. Additional bands of 148 kDa and 135 kDa were present in bovine isolate, compared to the reference isolate, while 152 kDa and 137 kDa bands were unique for caprine isolate. [Vet. World 2011; 4(10.000: 470-472

  19. Moist and dry heating-induced changes in protein molecular structure, protein subfractions, and nutrient profiles in camelina seeds.

    Peng, Quanhui; Khan, Nazir A; Wang, Zhisheng; Yu, Peiqiang

    2014-01-01

    The objectives of the present study were to investigate the nutritive value of camelina seeds (Camelina sativa L. Crantz) in ruminant nutrition and to use molecular spectroscopy as a novel technique to quantify the heat-induced changes in protein molecular structures in relation to protein digestive behavior in the rumen and intestine of dairy cattle. In this study, camelina seeds were used as a model for feed protein. The seeds were kept as raw (control) or heated in an autoclave (moist heating) or in an air-draft oven (dry heating) at 120°C for 60 min. The parameters evaluated were (1) chemical profiles, (2) Cornell Net Protein and Carbohydrate System protein subfractions, (3) nutrient digestibilities and estimated energy values, (4) in situ rumen degradation and intestinal digestibility, and (5) protein molecular structures. Compared with raw seeds, moist heating markedly decreased (52.73 to 20.41%) the content of soluble protein and increased (2.00 to 9.01%) the content of neutral detergent insoluble protein in total crude protein (CP). Subsequently, the rapidly degradable Cornell Net Protein and Carbohydrate System CP fraction markedly decreased (45.06 to 16.69% CP), with a concomitant increase in the intermediately degradable (45.28 to 74.02% CP) and slowly degradable (1.13 to 8.02% CP) fractions, demonstrating a decrease in overall protein degradability in the rumen. The in situ rumen incubation study revealed that moist heating decreased (75.45 to 57.92%) rumen-degradable protein and increased (43.90 to 82.95%) intestinal digestibility of rumen-undegradable protein. The molecular spectroscopy study revealed that moist heating increased the amide I-to-amide II ratio and decreased α-helix and α-helix-to-β-sheet ratio. In contrast, dry heating did not significantly change CP solubility, rumen degradability, intestinal digestibility, and protein molecular structures compared with the raw seeds. Our results indicated that, compared with dry heating, moist

  20. Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

    Sjöberg, Ronald; Mattsson, Cecilia; Andersson, Eni; Hellström, Cecilia; Uhlen, Mathias; Schwenk, Jochen M; Ayoglu, Burcu; Nilsson, Peter

    2016-09-25

    High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions. PMID:26417875

  1. Enzymatic hydrolysis of whey protein concentrates : peptide HPLC profiles

    Mota, M. V. T.; Ferreira, I. M. P. L. V. O.; Oliveira, M. B. P.; Rocha, Cristina M. R.; J. A. Teixeira; Torres, D; M. P. Gonçalves

    2004-01-01

    Hydrolysis of whey protein concentrates (WPCs) at different temperatures and pHs, using three enzymes: pepsin, trypsin, and Alcalase®, was monitored during more than 5 hr by reversed phase HPLC/UV, using a column containing a polystyrene-divinylbenzene copolymer-based packing, and an elution gradient from 8% to 80% acetonitrile containing 0.1% TFA. Peptides were separated according to their polarity and size, and degradation of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) ...

  2. Interactome profile of the host cellular proteins and the nonstructural protein 2 of porcine reproductive and respiratory syndrome virus.

    Li Wang

    Full Text Available The nonstructural protein 2 (NSP2 is considered to be one of crucial viral proteins in the replication and pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV. In the present study, the host cellular proteins that interact with the NSP2 of PRRSV were immunoprecipitated with anti-Myc antibody from the MARC-145 cells infected by a recombinant PRRSV with 3xMyc tag insertion in its NSP2-coding region, and then 285 cellular proteins interacting with NSP2 were identified by LC-MS/MS. The Gene Ontology and enriched KEGG Pathway bioinformatics analyses indicated that the identified proteins could be assigned to different subcellular locations and functional classes. Functional analysis of the interactome profile highlighted cellular pathways associated with infectious disease, translation, immune system, nervous system and signal transduction. Two interested cellular proteins-BCL2-associated athanogene 6 (BAG6 and apoptosis-inducing factor 1 (AIF1 which may involve in transporting of NSP2 to Endoplasmic reticulum (ER or PRRSV-driven apoptosis were validated by Western blot. The interactome data between PRRSV NSP2 and cellular proteins contribute to the understanding of the roles of NSP2 in the replication and pathogenesis of PRRSV, and also provide novel cellular target proteins for elucidating the associated molecular mechanisms of the interaction of host cellular proteins with viral proteins in regulating the viral replication.

  3. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences

    Meinicke Peter

    2009-01-01

    Abstract Background Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Description Based on machine learning techniques for Pfam domain detection, the UFO web ...

  4. Detecting protein candidate fragments using a structural alphabet profile comparison approach.

    Shen, Yimin; Picord, Géraldine; Guyon, Frédéric; Tuffery, Pierre

    2013-01-01

    Predicting accurate fragments from sequence has recently become a critical step for protein structure modeling, as protein fragment assembly techniques are presently among the most efficient approaches for de novo prediction. A key step in these approaches is, given the sequence of a protein to model, the identification of relevant fragments - candidate fragments - from a collection of the available 3D structures. These fragments can then be assembled to produce a model of the complete structure of the protein of interest. The search for candidate fragments is classically achieved by considering local sequence similarity using profile comparison, or threading approaches. In the present study, we introduce a new profile comparison approach that, instead of using amino acid profiles, is based on the use of predicted structural alphabet profiles, where structural alphabet profiles contain information related to the 3D local shapes associated with the sequences. We show that structural alphabet profile-profile comparison can be used efficiently to retrieve accurate structural fragments, and we introduce a fully new protocol for the detection of candidate fragments. It identifies fragments specific of each position of the sequence and of size varying between 6 and 27 amino-acids. We find it outperforms present state of the art approaches in terms (i) of the accuracy of the fragments identified, (ii) the rate of true positives identified, while having a high coverage score. We illustrate the relevance of the approach on complete target sets of the two previous Critical Assessment of Techniques for Protein Structure Prediction (CASP) rounds 9 and 10. A web server for the approach is freely available at http://bioserv.rpbs.univ-paris-diderot.fr/SAFrag. PMID:24303019

  5. Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

    LI Han; ZENG Qunli; WENG Yu; LU Deqiang; JIANG Huai; XU Zhengping

    2005-01-01

    Extremely Low Frequency Magnetic Fields (ELF MF) has been considered as a "possible human carcinogen" by International Agency for Research on Cancer (IARC) while credible mechanisms of its carcinogenicity remain unknown. In this study, a proteomics approach was employed to investigate the changes of protein expression profile induced by ELF MF in human breast cancer cell line MCF7, in order to determine ELF MF-responsive proteins. MCF7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h and the changes of protein profile were examined using two dimensional electrophoresis. Up to 6 spots have been statistically significantly altered (their expression levels were changed at least 5 fold up or down) compared with sham-exposed group. 19 ones were only detected in exposure group while 19 ones were missing. Three proteins were identified by LC-IT Tandem MS as RNA binding protein regulatory subunit、Proteasome subunit beta type 7 precursor and Translationally Controlled Tumor Protein. Our finding showed that 50 Hz, 0.4 mT ELF MF alternates the protein profile of MCF7 cell and may affect many physiological functions of normal cell and 2-DE coupled with MS is a promising approach to elucidating cellular effects of electromagnetic fields.

  6. Profil Protein Stadium Sporozoit Eimeria tenella Isolat Yogyakarta Melalui Analisis Protein SDS-PAGE (PROTEIN PROFILE OF THE SPOROZOITE OF Eimeria tenella ISOLATES FROM YOGYAKARTA USING SDS-PAGE PROTEIN ANALYSIS

    Galuh Tresnani

    2014-08-01

    Full Text Available Coccidiosis is one of the important diseases in poultry industry. In Indonesia the morbidity of thedisease is between 80 to 90%. A rapid and prompt diagnosis would be one of the essential steps ineradication and control of the disease. The objective of this study is to determine the protein profile ofsporozoite of Eimeria tenella isolated in Yogyakarta using sodium dodecyl sulphate polyacrilamide gelelectrophoresis (SDS-PAGE protein analysis. Protein analysis was performed in 12% polyacrilamide geland further electrophoresis at 100 volts and over-staining with Coomasie brilliant blue. The resultsshowed that the sporozoite of E. tenella isolated in Yogyakarta contained five proteins with molecularweights of 15, 26, 32, 80, and 91 kDa, respectively.

  7. dRHP-PseRA: detecting remote homology proteins using profile-based pseudo protein sequence and rank aggregation.

    Chen, Junjie; Long, Ren; Wang, Xiao-Long; Liu, Bin; Chou, Kuo-Chen

    2016-01-01

    Protein remote homology detection is an important task in computational proteomics. Some computational methods have been proposed, which detect remote homology proteins based on different features and algorithms. As noted in previous studies, their predictive results are complementary to each other. Therefore, it is intriguing to explore whether these methods can be combined into one package so as to further enhance the performance power and application convenience. In view of this, we introduced a protein representation called profile-based pseudo protein sequence to extract the evolutionary information from the relevant profiles. Based on the concept of pseudo proteins, a new predictor, called "dRHP-PseRA", was developed by combining four state-of-the-art predictors (PSI-BLAST, HHblits, Hmmer, and Coma) via the rank aggregation approach. Cross-validation tests on a SCOP benchmark dataset have demonstrated that the new predictor has remarkably outperformed any of the existing methods for the same purpose on ROC50 scores. Accordingly, it is anticipated that dRHP-PseRA holds very high potential to become a useful high throughput tool for detecting remote homology proteins. For the convenience of most experimental scientists, a web-server for dRHP-PseRA has been established at http://bioinformatics.hitsz.edu.cn/dRHP-PseRA/. PMID:27581095

  8. Texture profile in processed cheese: influence of the use of milk protein concentrates and whey protein concentrates

    Alisson Borges Souza

    2014-07-01

    Full Text Available The techno-functional properties of proteins related with the molecular characteristics are facilitated by partial unfolding of structures. From these interactions, the medium pH is presented as a major interferer in intensity and type of reaction that takes place. The intensity of denaturation and interaction of different proteins occur in different forms and intensity accordingly to the pH value of the medium in which they are located. This study aimed to verify the influence of interactions between whey protein concentrate/milk protein concentrate on the evolution of the texture profile of processed cheese at different pH values. We have analyzed samples of commercial whey protein concentrate (WPC and milk protein concentrate (MPC using 112.5g/kg processed cheese. The results were interpreted in terms of texture profile. It was also possible to optimize the different proportions of WPC and MPC, and pH value change the parameters of texture for creamy processed cheese and the pH was also an influencing factor in this optimization.

  9. Selectivity analysis of single binder assays used in plasma protein profiling

    Neiman, Maja; Fredolini, Claudia; Johansson, Henrik; Lehtiö, Janne; Nygren, Per-Åke; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2013-01-01

    The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries. PMID:24151238

  10. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter;

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affe...

  11. Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling

    Tome, Jacob M.; Ozer, Abdullah; Pagano, John M.; Gheba, Dan; Schroth, Gary P.; Lis, John T.

    2014-01-01

    RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its...

  12. Selectivity analysis of single binder assays used in plasma protein profiling

    Neiman, Maja; Fredolini, Claudia; Johansson, Henrik; Lehtiö, Janne; Nygren, Per-Åke; Uhlén, Mathias; Nilsson, Peter; Jochen M Schwenk

    2013-01-01

    The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from an...

  13. A Serum Protein Profile Predictive of the Resistance to Neoadjuvant Chemotherapy in Advanced Breast Cancers*

    Hyung, Seok-Won; Lee, Min Young; Yu, Jong-Han; Shin, Byunghee; Jung, Hee-Jung; Park, Jong-Moon; Han, Wonshik; Lee, Kyung-min; Moon, Hyeong-Gon; Zhang, Hui; Aebersold, Ruedi; Hwang, Daehee; Lee, Sang-Won; Yu, Myeong-Hee; Noh, Dong-Young

    2011-01-01

    Prediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to N...

  14. Hierarchical Partitioning of Metazoan Protein Conservation Profiles Provides New Functional Insights

    Witztum, Jonathan; Persi, Erez; Horn, David; Pasmanik-Chor, Metsada; Chor, Benny

    2014-01-01

    The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles). We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO) analysis tool, we explore functional enrichment of the “universal proteins”, those with homologues in al...

  15. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G.; Gelly, Jean-Christophe

    2016-01-01

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation —with Protein Blocks—, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the ‘Hard’ category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/. PMID:27319297

  16. Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation

    Aurora Moen

    2016-01-01

    Full Text Available Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007–2009. The new multiplex proximity extension assay (PEA technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase, epidermal growth factor (EGF; 142% increase, and monocyte chemotactic protein 4 (MCP-4; 70% increase. Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation.

  17. Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation.

    Moen, Aurora; Lind, Anne-Li; Thulin, Måns; Kamali-Moghaddam, Masood; Røe, Cecilie; Gjerstad, Johannes; Gordh, Torsten

    2016-01-01

    Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007-2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation. PMID:27293953

  18. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  19. HoxPred: automated classification of Hox proteins using combinations of generalised profiles

    Leyns Luc

    2007-07-01

    Full Text Available Abstract Background Correct identification of individual Hox proteins is an essential basis for their study in diverse research fields. Common methods to classify Hox proteins focus on the homeodomain that characterise homeobox transcription factors. Classification is hampered by the high conservation of this short domain. Phylogenetic tree reconstruction is a widely used but time-consuming classification method. Results We have developed an automated procedure, HoxPred, that classifies Hox proteins in their groups of homology. The method relies on a discriminant analysis that classifies Hox proteins according to their scores for a combination of protein generalised profiles. 54 generalised profiles dedicated to each Hox homology group were produced de novo from a curated dataset of vertebrate Hox proteins. Several classification methods were investigated to select the most accurate discriminant functions. These functions were then incorporated into the HoxPred program. Conclusion HoxPred shows a mean accuracy of 97%. Predictions on the recently-sequenced stickleback fish proteome identified 44 Hox proteins, including HoxC1a only found so far in zebrafish. Using the Uniprot databank, we demonstrate that HoxPred can efficiently contribute to large-scale automatic annotation of Hox proteins into their paralogous groups. As orthologous group predictions show a higher risk of misclassification, they should be corroborated by additional supporting evidence. HoxPred is accessible via SOAP and Web interface http://cege.vub.ac.be/hoxpred/. Complete datasets, results and source code are available at the same site.

  20. A Click Chemistry‐Based Proteomic Approach Reveals that 1,2,4‐Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile

    Ismail, Hanafy M.; Barton, Victoria E.; Panchana, Matthew; Charoensutthivarakul, Sitthivut; Biagini, Giancarlo A.; Ward, Stephen A.

    2016-01-01

    Abstract In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross‐resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4‐trioxolane activity based protein‐profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi‐quantitatively, suggesting a common mechanism of action that raises concerns about potential cross‐resistance liabilities. PMID:27397940

  1. Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

    Bhagyawant, S S; Gupta, N; Shrivastava, N

    2015-01-01

    The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro. PMID:26516115

  2. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  3. Characterization of tomato germplasm through seeds through rage protein profiling by sds-page

    The 24 tomato genotypes, including 5 hybrids (Avinash-II, CKD-1092, CKD-1093, CKD-1695, CKD-1088), seven germplasm lines (07039, 09091, 27-07, 42-07, 07011, 09076, 09078) and twelve cultivars (Excellence, Nagina, Naqeeb, Advanta-1202, PTM-1431, Pakit, Rio Grande, Lyp-1, Roma, Continental, VCT-I, Peto-86), were analyzed by SDS-PAGE for total soluble seed storage proteins on 10% gels. A low level of variability was observed in protein profiles of tomato genotypes. Dendrogram based on electrophoretic data clustered the 24 genotypes in four major groups. All germplasm lines illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. However, among tomato cultivars, VCT-l found to be the most divergent and could be distinguished from others on the basis of two peptides i.e. 58 kDa and 15 kDa. Similarly, in case of hybrids, a peptide of 58 kDa was absent in Avinash while present in all other hybrids. Another peptide of 64 kDa was unique to Avinash and absent in all other hybrids. Therefore, among hybrids, Avinash can be distinguished from others based on these peptide differences. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in tomato. Among 42 resolved peptides, eleven could be identified from databases. On the basis of molecular weight similarity, identified peptides were SSP-83 as Alkaline alpha-galactosidase, SSP-78 as BiP, SSP-66 as vicilin, SSP-64 as DELLA Protein, SSP-58 as SNF1, SSP-41 as SNF4, SSP-36 as putative galactinol synthase 1, SSP-33 as Xyloglucan endotransg-lycosylases, SSP-26 as expansin family, SSP-10 as Putative vicilin and SSP-8 as albumin protein. In conclusion, seed storage profiling by SDS-PAGE can economically be used to asses the genetic variation in different tomato genotypes. (author)

  4. Protein profiles of field isolates ofBacillus anthracis from different endemic areas of Indonesia

    M Bhakti Poerwadikarta

    1998-03-01

    Full Text Available Sonicated cell-free extract proteins of 14 field isolates ofBacillus anthracis from six different endemic areas of Indonesia were analyzed by the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE methods . The protein profiles of each field isolate tested demonstrated slightly different at the protein bands with molecular weights of 18, 37, 52, 65 and 70 kDa, and varied between the field isolates and vaccine strains. The variation could provide clues to the source of anthrax transmission whether it was originated from similar strain or not.

  5. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  6. How the diffusivity profile reduces the arbitrariness of protein folding free energies

    Hinczewski, Michael; Dzubiella, Joachim; Netz, Roland R

    2010-01-01

    The concept of a protein diffusing in its free energy folding landscape has been fruitful for both theory and experiment. Yet the choice of the reaction coordinate (RC) introduces an undesirable degree of arbitrariness into the problem. We analyze extensive simulation data of an alpha-helix in explicit water solvent as it stochastically folds and unfolds. The free energy profiles for different RCs exhibit significant variation, some having an activation barrier, others not. We show that this variation has little effect on the predicted folding kinetics if the diffusivity profiles are properly taken into account. This kinetic quasi-universality is rationalized by an RC rescaling, which, due to the reparameterization invariance of the Fokker-Planck equation, allows the combination of free energy and diffusivity effects into a single function, the rescaled free energy profile. This rescaled free energy indeed shows less variation among different RCs than the bare free energy and diffusivity profiles separately d...

  7. A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells

    Lehtiö Janne

    2009-11-01

    Full Text Available Abstract Background B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL is presumed to derive predominantly from naïve, pre-germinal centre (pre-GC B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods Subpopulations of B cells representing the germinal centre (GC, the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS. Results Quantitative MS data of significant protein peaks (p-value Conclusion AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.

  8. Major urinary protein (MUP) profiles show dynamic changes rather than individual ‘barcode’ signatures

    Thoß, M.; Luzynski, K.C.; Ante, M.; Miller, I.; Penn, D.J.

    2016-01-01

    House mice (Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous (‘minor’) bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis). PMID:26973837

  9. Major urinary protein (MUP profiles show dynamic changes rather than individual ‘barcode’ signatures

    Michaela eThoß

    2015-06-01

    Full Text Available House mice (Mus musculus produce a variable number of major urinary proteins (MUPs, and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major bands were surprisingly homogenous (and hence most MUPs are not polymorphic, but we also found inconspicuous (‘minor’ bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age, and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis.

  10. MBPpred: Proteome-wide detection of membrane lipid-binding proteins using profile Hidden Markov Models.

    Nastou, Katerina C; Tsaousis, Georgios N; Papandreou, Nikos C; Hamodrakas, Stavros J

    2016-07-01

    A large number of modular domains that exhibit specific lipid binding properties are present in many membrane proteins involved in trafficking and signal transduction. These domains are present in either eukaryotic peripheral membrane or transmembrane proteins and are responsible for the non-covalent interactions of these proteins with membrane lipids. Here we report a profile Hidden Markov Model based method capable of detecting Membrane Binding Proteins (MBPs) from information encoded in their amino acid sequence, called MBPpred. The method identifies MBPs that contain one or more of the Membrane Binding Domains (MBDs) that have been described to date, and further classifies these proteins based on their position in respect to the membrane, either as peripheral or transmembrane. MBPpred is available online at http://bioinformatics.biol.uoa.gr/MBPpred. This method was applied in selected eukaryotic proteomes, in order to examine the characteristics they exhibit in various eukaryotic kingdoms and phyla. PMID:27048983

  11. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  12. Evolution of liquid holdup profile in a standing protein stabilized foam.

    Wang, Zebin; Narsimhan, Ganesan

    2004-12-01

    Evolution of liquid holdup profile in a standing foam formed by whipping and stabilized by sodium caseinate in the presence of xanthan gum when subjected to 16 and 29g centrifugal force fields was measured using magnetic resonance imaging for different pH, ionic strength, protein and xanthan gum concentrations. Drainage resulted in the formation of a separate liquid layer at the bottom at longer times. Foam drainage was slowest at pH 7, lower ionic strength, higher protein and gum concentrations. Foam was found to be most stable at pH 5.1 near the isoelectric point of protein, lower ionic strength and higher protein and xanthan gum concentrations. A predicted equilibrium liquid holdup profile based on a previous model (G. Narsimhan, J. Food Eng. 14 (1991) 139) agreed well with experimental values at sufficiently long times. A proposed model for velocity of drainage of a power law fluid in a Plateau border for two different simplified geometries was incorporated in a previously developed model for foam drainage (G. Narsimhan, J. Food Eng. 14 (1991) 139) to predict the evolution of liquid holdup profiles. The model predictions for simplified circular geometry of Plateau border compared well with the experimental data of liquid holdup profiles at small times. At longer times, however, the predicted liquid holdup profile was larger than the observed, this discrepancy being due to coarsening of bubble size and decrease in foam height not accounted for in the model. A Newtonian model for foam drainage under predicted drainage rates did not agree with the experimental data. PMID:15476794

  13. Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

    Nicholas, H B; Persson, B; Jörnvall, H; Hempel, J.

    1995-01-01

    The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same c...

  14. Biochemical composition and protein profile of alpaca (Vicugna pacos) oviductal fluid.

    Apichela, S A; Argañaraz, M E; Zampini, R; Vencato, J; Miceli, D C; Stelletta, C

    2015-03-01

    Knowledge and assessment of the constituents of the oviductal fluid (OF) in camelids is necessary for a correct formulation of specific culture media for the development of reproductive biotechnology. This study is the first describing the biochemical composition and SDS-PAGE protein profile of alpaca oviductal fluid in non-pregnant animals and animals that have completed the first month and second month of gestation. Samples were also classified into oviducts that were ipsilateral or contralateral to the ovary with corpus luteum. No differences were found between both oviducts, whereas pregnant and non-pregnant females displayed significant differences in the biochemical composition and protein profile of the oviductal fluid. Relative albumin content was higher in non-pregnant females. Relative creatinine content in OF from females that have completed the second month of gestation was lower than non-pregnant females and females that have completed the first month of gestation. Ion Na(+) concentration was higher in OF from non-pregnant females when compared with pregnant ones. The protein profile of non-pregnant females showed five protein bands of 70, 42, 25, 24 and 19kDa that were significantly more intense compared with pregnant animals. Bands were identified as moesin, actin cytoplasmic 2, hydroxypyruvate isomerase, ferritin light chain and peroxiredoxin-6 with MALDI/MS. Our results encourage more thorough future studies, in order to unravel the complex reproductive processes of the South American camelid oviduct. PMID:25592861

  15. A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

    Christina Marie Gutierrez

    2015-11-01

    Full Text Available Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk versus late/nulliparity (high risk. Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

  16. Differential protein profiling as a potential multi-marker approach for TSE diagnosis

    Hogarth Caroline

    2009-11-01

    Full Text Available Abstract Background Transmissible spongiform encephalopathy describes a family of diseases affecting both man and animals. Current tests for the diagnosis of these diseases are based on the detection of an abnormal misfolded form of the host protein PrP which is found within the central nervous and lymphoreticular systems of affected animals. Recently, concern that this marker may not be as reliable as previously thought, coupled with an urgentneed for a pre-clinical live animal test, has led to the search for alternative assays for the detection of TSE disease. Methods This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers. Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. Results Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. Conclusion Differential protein expression profiling has the potential to be used for the detection of disease in TSE

  17. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology. PMID:26945504

  18. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

    Ismail, Hanafy M; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

    2016-01-01

    The mechanism of action of the artemisinin (ART) class of antimalarial drugs, the most important antimalarial drug class in use today, remains controversial, despite more than three decades of intensive research. We have developed an unbiased chemical proteomic approach using a suite of ART activity-based protein profiling probes to identify proteins within the malaria parasite that are alkylated by ART, including proteins involved in glycolysis, hemoglobin metabolism, and redox defense. The ...

  19. SDS-Page Seed Storage Protein Profiles in Chili Peppers (Capsicum L.

    Owk ANIEL KUMAR

    2010-09-01

    Full Text Available Seed protein banding patterns (SDS-PAGE were studied from eighteen genotypes of chili pepper (Capsicum L. A total of 21 protein polypeptide bands with molecular weight ranging from 18.6 to 72.0 kD were recorded. Among the genotypes �CA18�, �CA21� and �CA27� represented maximum number of protein bands (12. Band no. (11 and (5,12 are exclusive to C. annuum L. and C. frutescens L. genotypes respectively. Average percent similarity was highest (100% between �CA2� and �CA8� genotypes and the UPGMA dendrogram represented low genetic diversity. The study revealed that considerable intra and inter-specific differences were found in the genotypes. The variability of protein profiles in the genotypes suggested that these selected genotypes can be a good source for crop improvement through hybridization programs.

  20. Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

    Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigne-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

    2013-12-01

    Development of alternative, non-petroleum based sources of bioenergy that can be applied in the short-term find great promise in the use of highly abundant and renewable lignocellulosic plant biomass.1 This material obtained from different feedstocks, such as forest litter or agricultural residues, can yield liquid fuels and other chemical products through biorefinery processes.2 Biofuels are obtained from lignocellulosic materials by chemical pretreatment of the biomass, followed by enzymatic decomposition of cellulosic and hemicellulosic compounds into soluble sugars that are converted to desired chemical products via microbial metabolism and fermentation.3, 4 To release soluble sugars from polymeric cellulose multiple enzymes are required, including endoglucanase, exoglucanase, and β-glucosidase.5, 6 However, the enzymatic hydrolysis of cellulose into soluble sugars remains a significant limiting factor to the efficient and economically viable utilization of lignocellulosic biomass for transport fuels.7, 8 The primary industrial source of cellulose and hemicellulases is the mesophilic soft-rot fungus Trichoderma reesei,9 having widespread applications in food, feed, textile, pulp, and paper industries.10 The genome encodes 200 glycoside hydrolases, including 10 cellulolytic and 16 hemicellulolytic enzymes.11 The hypercellulolytic catabolite derepressed strain RUT-C30 was obtained through a three-step UV and chemical mutagenesis of the original T. reesei strain QM6a,12, 13 in which strains M7 and NG14 were intermediate, having higher cellulolytic activity than the parent strain but less activity and higher catabolite repression than RUT-C30.14 Numerous methods have been employed to optimize the secreted enzyme cocktail of T. reesei including cultivation conditions, operational parameters, and mutagenesis.3 However, creating an optimal and economical enzyme mixture for production-scale biofuels synthesis may take thousands of experiments to identify.

  1. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    Higa, A.M.; Noronha, M.D.N. [Universidade do Estado do Amazonas (UEA), Manaus, AM (Brazil). Rede Proteomica do Amazonas (Proteam). Lab. de Genomica e Proteomica; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L. [Universidade Federal do Amazonas (UFAM), Manaus, AM (Brazil). Pos-Graduacao em Biotecnologia

    2008-07-01

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with {approx} 60, 70 and 80 kDa were detected in gel acidic region with pI {approx} 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI {approx} 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with {approx} 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI {approx} 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course.

  2. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with ∼ 60, 70 and 80 kDa were detected in gel acidic region with pI ∼ 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI ∼ 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with ∼ 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI ∼ 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course

  3. Proteomic profile of carbonylated proteins in rat liver: discovering possible mechanisms for tetracycline-induced steatosis.

    Deng, Zhenglu; Yan, Siyu; Hu, Hui; Duan, Zhigui; Yin, Lanxuan; Liao, Shenke; Sun, Yubai; Yin, Dazhong; Li, Guolin

    2015-01-01

    To investigate biochemical mechanisms for the tetracycline-induced steatosis in rats, targeted proteins of oxidative modification were profiled. The results showed that tetracycline induced lipid accumulation, oxidative stress, and cell viability decline in HepG2 cells only under the circumstances of palmitic acid overload. Tetracycline administration in rats led to significant decrement in blood lipids, while resulted in more than four times increment in intrahepatic triacylglycerol and typical microvesicular steatosis in the livers. The triacylglycerol levels were positively correlated with oxidative stress. Proteomic profiles of carbonylated proteins revealed 26 targeted proteins susceptible to oxidative modification and most of them located in mitochondria. Among them, the long-chain specific acyl-CoA dehydrogenase was one of the key enzymes regulating fatty acid β-oxidation. Oxidative modification of the enzyme in the tetracycline group depressed its enzymatic activity. In conclusion, the increased influx of lipid into the livers is the first hit of tetracycline-induced microvesicular steatosis. Oxidative stress is an essential part of the second hit, which may arise from the lipid overload and attack a series of functional proteins, aggravating the development of steatosis. The 26 targeted proteins revealed here provide a potential direct link between oxidative stress and tetracycline-induced steatosis. PMID:25332112

  4. Transcriptional profiling of protein expression related genes of Pichia pastoris under simulated microgravity.

    Feng Qi

    Full Text Available The physiological responses and transcription profiling of Pichia pastoris GS115 to simulated microgravity (SMG were substantially changed compared with normal gravity (NG control. We previously reported that the recombinant P. pastoris grew faster under SMG than NG during methanol induction phase and the efficiencies of recombinant enzyme production and secretion were enhanced under SMG, which was considered as the consequence of changed transcriptional levels of some key genes. In this work, transcriptiome profiling of P. pastoris cultured under SMG and NG conditions at exponential and stationary phases were determined using next-generation sequencing (NGS technologies. Four categories of 141 genes function as methanol utilization, protein chaperone, RNA polymerase and protein transportation or secretion classified according to Gene Ontology (GO were chosen to be analyzed on the basis of NGS results. And 80 significantly changed genes were weighted and estimated by Cluster 3.0. It was found that most genes of methanol metabolism (85% of 20 genes and protein transportation or secretion (82.2% of 45 genes were significantly up-regulated under SMG. Furthermore the quantity and fold change of up-regulated genes in exponential phase of each category were higher than those of stationary phase. The results indicate that the up-regulated genes of methanol metabolism and protein transportation or secretion mainly contribute to enhanced production and secretion of the recombinant protein under SMG.

  5. Soy Germ Protein With or Without-Zn Improve Plasma Lipid Profile in Metabolic Syndrome Women

    SIWI PRAMATAMA MARS WIJAYANTI

    2012-03-01

    Full Text Available The aim of this research was to determine the effect of soy germ protein on lipid profile of metabolic syndrome (MetS patients. Respondents were 30 women with criteria, i.e. blood glucose level > normal, body mass index > 25 kg/m2, hypertriglyceridemia, low cholesterol-HDL level, 40-65 years old, living in Purwokerto, and signed the informed consent. The project was approved by the ethics committee of the Medical Faculty from Gadjah Mada University-Yogyakarta. Respondents were divided into three randomly chosen groups consisting of ten women each. The first, second, and third groups were treated, respectively, with milk enriched soy germ protein plus Zn, milk enriched soy germ protein (without Zn, and placebo for two months. Blood samples were taken at baseline, one and two months after observation. Two months after observation the groups consuming milk enriched with soy germ protein, both with or without Zn, had their level of cholesterol-total decrease from 215.8 to 180.2 mg/dl (P = 0.03, triglyceride from 240.2 to 162.5 mg/dl (P = 0.02, and LDL from 154.01 to 93.85 mg/dl (P = 0.03. In contrast, HDL increased from 38.91 to 49.49 mg/dl (P = 0.0008. In conclusion, soy germ protein can improve lipid profile, thus it can inhibit atherosclerosis incident.

  6. PPINGUIN: Peptide Profiling Guided Identification of Proteins improves quantitation of iTRAQ ratios

    Bauer, Chris; Kleinjung, Frank; Rutishauser, Dorothea; Panse, Christian; Chadt, Alexandra; Dreja, Tanja; Al-Hasani, Hadi; Reinert, Knut; Schlapbach, Ralph; Schuchhardt, Johannes

    2012-01-01

    BACKGROUND: Recent development of novel technologies paved the way for quantitative proteomics. One of the most important among them is iTRAQ, employing isobaric tags for relative or absolute quantitation. Despite large progress in technology development, still many challenges remain for derivation and interpretation of quantitative results. One of these challenges is the consistent assignment of peptides to proteins. RESULTS: We have developed Peptide Profiling Guided Identification of Pr...

  7. ACUTE PHASE PROTEINS, LIPID PROFILE AND PROINFLAMMATORY CYTOKINES IN HEALTHY AND BRONCHOPNEUMONIC WATER BUFFALO CALVES

    El-Bahr, Sabry M.; Wael M. EL-Deeb

    2013-01-01

    The aim of the present study was to evaluate the diagnostic value of Acute Phase Proteins (APP), lipid profiles and proinflammatory cytokines in healthy and bronchopneumonic water buffalo calves. Therefore, sixty water buffalo calves (9±1 month old, 175±15 kg) were divided into two equal groups, the first group represented healthy, control, calves whereas calves of the second group were affected with bronchopneumonia. Total leukocytic and differential counts were determined. Serum total pr...

  8. Changes in protein profiles and isoelectric points of different Candida species in response to antifungal agents

    Šalplachta, Jiří; Horká, Marie; Růžička, F.; Vykydalová, Marie; Kubesová, Anna; Šlais, Karel

    Universidad de La Laguna, 2013. s. 127. [International Symposium on Electro- and Liquid Phase-separation Techniques /20./. 06.10.2013-09.10.2013, Puerto de la Cruz, Tenerife] R&D Projects: GA MV VG20102015023; GA MZd(CZ) MZd 9678-4 Institutional support: RVO:68081715 Keywords : capillary isoelectric focusing and MALDI-TOF MS * protein profile * Candida species Subject RIV: CB - Analytical Chemistry, Separation

  9. Serum protein profiles as potential biomarkers for infectious disease status in pigs

    Koene Miriam GJ; Mulder Han A; Stockhofe-Zurwieden Norbert; Kruijt Leo; Smits Mari A

    2012-01-01

    Abstract Background In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circ...

  10. Altered brain protein expression profiles are associated with molecular neurological dysfunction in the PKU mouse model

    Imperlini, Esther; Orrù, Stefania; Corbo, Claudia; Daniele, Aurora; Salvatore, Francesco

    2014-01-01

    Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU-associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury-PahEnu2 mice (a mouse model of...

  11. Protein profiling reveals consequences of lifestyle choices on predicted biological aging

    Stefan Enroth; Sofia Bosdotter Enroth; Åsa Johansson; Ulf Gyllensten

    2015-01-01

    Ageing is linked to a number of changes in how the body and its organs function. On a molecular level, ageing is associated with a reduction of telomere length, changes in metabolic and gene-transcription profiles and an altered DNA-methylation pattern. Lifestyle factors such as smoking or stress can impact some of these molecular processes and thereby affect the ageing of an individual. Here we demonstrate by analysis of 77 plasma proteins in 976 individuals, that the abundance of circulatin...

  12. Prediction of mitochondrial protein function by comparative physiology and phylogenetic profiling.

    Cheng, Yiming; Perocchi, Fabiana

    2015-01-01

    According to the endosymbiotic theory, mitochondria originate from a free-living alpha-proteobacteria that established an intracellular symbiosis with the ancestor of present-day eukaryotic cells. During the bacterium-to-organelle transformation, the proto-mitochondrial proteome has undergone a massive turnover, whereby less than 20 % of modern mitochondrial proteomes can be traced back to the bacterial ancestor. Moreover, mitochondrial proteomes from several eukaryotic organisms, for example, yeast and human, show a rather modest overlap, reflecting differences in mitochondrial physiology. Those differences may result from the combination of differential gain and loss of genes and retargeting processes among lineages. Therefore, an evolutionary signature, also called "phylogenetic profile", could be generated for every mitochondrial protein. Here, we present two evolutionary biology approaches to study mitochondrial physiology: the first strategy, which we refer to as "comparative physiology," allows the de novo identification of mitochondrial proteins involved in a physiological function; the second, known as "phylogenetic profiling," allows to predict protein functions and functional interactions by comparing phylogenetic profiles of uncharacterized and known components. PMID:25631025

  13. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  14. Combinatorial modeling of protein folding kinetics: free energy profiles and rates

    Henry, Eric R. [Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 104, Bethesda, MD 20892-0520 (United States); Eaton, William A. [Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Room 104, Bethesda, MD 20892-0520 (United States)]. E-mail: eaton@helix.nih.gov

    2004-12-27

    A combinatorial approach has been used to determine the optimal assumptions and robustness of simple statistical mechanical models for protein folding. By combining alternative plausible assumptions and different enumeration schemes for constructing partition functions, 76 closely related Ising-like models were generated. These include various contiguous sequence approximations, the possibility of forming a disordered loop between ordered segments, the use of an atomistic or coarse-grained representation of the protein structure, and the choice of ordered residues or native contacts as a reaction coordinate. We also consider all 2{sup N} conformations of an N-residue protein (e.g., 10{sup 30} conformations for a 100-residue protein). Although the number of configurations is much too large to list, a free energy profile can be calculated using a build-up procedure by accumulation and recombination of partial partition functions. The predictions of the 76 models were tested against two kinds of experimental data - folding rates and the determination of two-state behavior for 25 proteins. Two-state behavior was judged by the relative magnitude of the dominant relaxation calculated from the rate matrix for motion on the free energy profile. The relative performance of each assumption was evaluated using rank sum statistics which show, with the exception of the single sequence approximation, that this class of models is not sensitive to alternative assumptions. Two-state free energy profiles are calculated for almost all but the {alpha}-helical proteins, and surprisingly accurate rates are predicted in both the absence and presence of denaturant. The combinatorics also show that an {alpha}-carbon representation of the protein structure does nearly as well as atomistic descriptions, possibly reflecting partial interatomic interactions between native residues in structures of the transition state ensemble. With its coarse-grained description of both the energy and entropy

  15. Protein profile study of Pap smear and tissue of cervix by high performance liquid chromatography: laser induced fluorescence

    Sujatha, N.; Rai, Lavanya; Kumar, Pratap; Krishnanand, B. R.; Mahato, K. K.; George, Sajan D.; Kartha, V. B.; C, Santhosh

    2007-02-01

    HPLC combined with laser induced fluorescence provides a very sensitive method for the separation and identification of the many proteins present in clinical samples. Protein profiles of clinical samples like Pap smear and tissue samples, from subjects with cervical cancer and normal volunteers, were recorded using HPLC-LIF. The protein profiles were analyzed by Principal Component Analysis (PCA). The profiles were characterized by parameters like scores of the factors, sum of squared residuals, and Mahalanobis Distance, derived from PCA. Parameters of each sample were compared with those of a standard set and Match/ No Match results were generated. Good discrimination between normal and malignant samples was achieved with high sensitivity and specificity.

  16. Comparison of antioxidant capacity, protein profile and carbohydrate content of whey protein fractions.

    Önay-Uçar, Evren; Arda, Nazlı; Pekmez, Murat; Yılmaz, Ayşe Mine; Böke-Sarıkahya, Nazlı; Kırmızıgül, Süheyla; Yalçın, A Suha

    2014-05-01

    Whey is used as an additive in food industry and a dietary supplement in nutrition. Here we report a comparative analysis of antioxidant potential of whey and its fractions. Fractions were obtained by size exclusion chromatography, before and after enzymatic digestion with pepsin or trypsin. Superoxide radical scavenging, lipid peroxidation inhibition and cupric ion reducing activities of different fractions were checked. Peptides were detected by SDS-PAGE and GC-MS was used to determine carbohydrate content of the fractions. All samples showed antioxidant activity and the second fraction of the trypsin hydrolysate showed the highest superoxide radical scavenging activity. CUPRAC value of this fraction was two-times higher than that of whey filtrate. The first fraction of the pepsin hydrolysate was the most effective inhibitor of lipid peroxidation. Each sample exhibited a different polypeptide profile. Different percentages of carbohydrates were identified in whey filtrate and in all second fractions, where galactose was the major component. PMID:24360416

  17. Comparative protein profiles: potential molecular markers from spermatozoa of Acipenseriformes (Chondrostei, Pisces).

    Li, Ping; Hulak, Martin; Rodina, Marek; Sulc, Miroslav; Li, Zhi-Hua; Linhart, Otomar

    2010-12-01

    Sturgeon and paddlefish (Acipenseriformes), the source of roe consumed as caviar, are a unique and commercially valuable group of ancient fishes. In this study, comparative proteomics was used to analyze protein profiles of spermatozoa from five sturgeon species and one paddlefish: Siberian sturgeon (Acipenser baerii), sterlet (A. ruthenus), Russian sturgeon (A. gueldenstaedtii), starry sturgeon (A. stellatus), beluga (Huso huso), and Mississippi paddlefish (Polyodon spathula). Protein profiles of spermatozoa were determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) high-resolution gels. The peptides, previously selected by 2-DE analysis as potentially species-specific, were obtained by "in-gel" tryptic digestion, followed by matrix-associated laser desorption/ionization time-of-flight/mass spectrometry (MALDI-TOF/MS). Among the 23 protein spots selected, 14 were identified as isoforms of enolase B present in all species, but with different isoelectric points or molecular mass. Exceptions were A. ruthenus and H. huso, species with a close phylogenetic relationship. Glycerol-3-phosphate dehydrogenase was detected exclusively in P. spathula. Phosphoglycerate kinase was detected only in A. ruthenus and H. huso, and 3 additional proteins (fructose bisphosphate aldolase A-2, glycogen phosphorylase type IV and glyceraldehyde-3-phosphate dehydrogenase) were found exclusively in A. gueldenstaedtii and H. huso. This study points to the application of proteomics for differential characterization and comparative studies of acipenseriform species at the molecular level. PMID:20869341

  18. Transcriptional Profiling of a Selective CREB Binding Protein Bromodomain Inhibitor Highlights Therapeutic Opportunities.

    Chekler, Eugene L Piatnitski; Pellegrino, Jessica A; Lanz, Thomas A; Denny, R Aldrin; Flick, Andrew C; Coe, Jotham; Langille, Jonathan; Basak, Arindrajit; Liu, Shenping; Stock, Ingrid A; Sahasrabudhe, Parag; Bonin, Paul D; Lee, Kevin; Pletcher, Mathew T; Jones, Lyn H

    2015-12-17

    Bromodomains are involved in transcriptional regulation through the recognition of acetyl lysine modifications on diverse proteins. Selective pharmacological modulators of bromodomains are lacking, although the largely hydrophobic nature of the pocket makes these modules attractive targets for small-molecule inhibitors. This work describes the structure-based design of a highly selective inhibitor of the CREB binding protein (CBP) bromodomain and its use in cell-based transcriptional profiling experiments. The inhibitor downregulated a number of inflammatory genes in macrophages that were not affected by a selective BET bromodomain inhibitor. In addition, the CBP bromodomain inhibitor modulated the mRNA level of the regulator of G-protein signaling 4 (RGS4) gene in neurons, suggesting a potential therapeutic opportunity for CBP inhibitors in the treatment of neurological disorders. PMID:26670081

  19. Peranan Faktor Imun dan Profil Protein dalam Penelitian dan Pengembangan Vaksin Malaria Iradiasi

    Mukh Syaifudin

    2014-03-01

    Full Text Available Pengembangan vaksin terhadap malaria yang merupakan penyakit yang mematikan tetap menjadi satu prioritas kesehatan masyarakat global, termasuk pemanfaatan parasit iradiasi sebagai bahan vaksin. Imunisasi dengan sporozoit iradiasi mampu memberikan imunitas protektif pada hewan coba dan sukarelawan. Mekanisme sistem kekebalan tubuh ini banyak dipelajari karena merupakan faktor penting dalam pengembangan vaksin, demikian halnya profil dan/atau ekspresi protein pasca iradiasi yang terkait erat dengan keamanan dan aspek lain dari bahan vaksin. Meskipun telah melalui penelitian yang ekstensif, vaksin yang aman dan protektif belum dapat diperoleh karena masih diperlukan pengetahuan yang lebih mendalam mengenai mekanisme imunitas dan protein dalam litbang malaria. Hasil penelitian menunjukkan bahwa sel limfosit T berperan penting dalam pengaturan respon imun dan pembentukan memori imunologik yang mengontrol dan mengeliminasi infeksi. Sitokin proinflamasi seperti interleukin-12 (IL-12, interferon-gamma (IFN-γ, dan tumor necrosis factor alpha (TNF-α juga merupakan mediator esensial dari imunitas protektif pada malaria eritrositik. Berbagai pendekatan lain terkait respon imun seperti genetika molekuler saat ini sedang dilakukan. Studi juga menunjukkan bahwa profil protein bergantung pada beberapa faktor yang akan dibahas lebih lanjut dalam makalah. The development of vaccine against malaria as the deadly disease remains the global public health priority; and it includes the use of irradiated parasites as vaccine materials. Immunization with irradiated sporozoites could provide protective immunity in animals and volunteers. The mechanism of this body immunity system has been studied widely due to its important role in the development of vaccines and profiles and/or protein expression post-irradiation which are closely related to safety and other aspects of vaccine materials. Even though extensive research has been done, a safe and protective vaccine

  20. Qualitative and Quantitative Changes in Protein Profile of Various Tissue of Tropical Tasar Silkworm, Antheraea mylitta Drury

    P.K. Mishra

    2011-01-01

    Full Text Available In the present study, quantitative and qualitative changes in protein profile of different tissue of larvae, pupae, adult and eggs of Tasar silkworm Antheraea mylitta Drury was investigated. Stage and age dependent variation in protein concentration and SDS-PAGE protein profile of 36 and 64 kDa protein was observed in different tissue. The concentration of protein was recorded higher in eggs laid by fresh moth than 3 days old moth and significant variation was also noticed in normal and depressed eggs. Interestingly, substantial changes in SDS-PAGE protein profile was observed in normal and depressed eggs and eggs laid by fresh moth than 3 days old moth. Haemolymph and midgut protein concentration was recorded higher in 3rd and 5th instar feeding larvae and in 4th instar mature larvae. Concentration of protein in the haemolymph of pupae before the brain window becomes opaque was higher in both the sexes than opaque stage. Fat body protein concentration in larvae showed increasing trend from 3rd to 5th instar larvae and it was higher in pupae after the brain window becomes opaque and fresh moth. In addition, higher protein concentration was recorded in gonads of pupae after the brain window becomes opaque and in reproductive organs of fresh moth. Present findings would promote to further understand the precise reason for depression of eggs and changes in protein profile in different tissue of A. mylitta.

  1. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences

    Meinicke Peter

    2009-09-01

    Full Text Available Abstract Background Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Description Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. Conclusion For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

  2. Altered brain protein expression profiles are associated with molecular neurological dysfunction in the PKU mouse model.

    Imperlini, Esther; Orrù, Stefania; Corbo, Claudia; Daniele, Aurora; Salvatore, Francesco

    2014-06-01

    Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU-associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury-PahEnu2 mice (a mouse model of PKU). We compared the cerebral protein expression of homozygous PKU mice with that of their heterozygous counterparts using two-dimensional difference gel electrophoresis analysis, and identified 21 differentially expressed proteins, four of which were over-expressed and 17 under-expressed. An in silico bioinformatic approach indicated that protein under-expression was related to neuronal differentiation and dendritic growth, and to such neurological disorders as progressive motor neuropathy and movement disorders. Moreover, functional annotation analyses showed that some identified proteins were involved in oxidative metabolism. To further investigate the proteins involved in the neurological damage, we validated two of the proteins that were most strikingly under-expressed, namely, Syn2 and Dpysl2, which are involved in synaptic function and neurotransmission. We found that Glu2/3 and NR1 receptor subunits were over-expressed in PKU mouse brain. Our results indicate that differential expression of these proteins may be associated with the processes underlying PKU brain dysfunction, namely, decreased synaptic plasticity and impaired neurotransmission. We identified a set of proteins whose expression is affected by hyperphenylalaninemia. We think that phenylketonuria (PKU) brain dysfunction also depends on reduced Syn2 and Dpysl2 levels, increased Glu2/3 and NR1 levels, and decreased Pkm, Ckb, Pgam1 and Eno1 levels. These findings finally confirm that alteration in synaptic

  3. Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

    Cardona-Correa, Albin; Rios-Velazquez, Carlos

    2016-05-01

    The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents. PMID

  4. PPINGUIN: Peptide Profiling Guided Identification of Proteins improves quantitation of iTRAQ ratios

    Bauer Chris

    2012-02-01

    Full Text Available Abstract Background Recent development of novel technologies paved the way for quantitative proteomics. One of the most important among them is iTRAQ, employing isobaric tags for relative or absolute quantitation. Despite large progress in technology development, still many challenges remain for derivation and interpretation of quantitative results. One of these challenges is the consistent assignment of peptides to proteins. Results We have developed Peptide Profiling Guided Identification of Proteins (PPINGUIN, a statistical analysis workflow for iTRAQ data addressing the problem of ambiguous peptide quantitations. Motivated by the assumption that peptides uniquely derived from the same protein are correlated, our method employs clustering as a very early step in data processing prior to protein inference. Our method increases experimental reproducibility and decreases variability of quantitations of peptides assigned to the same protein. Giving further support to our method, application to a type 2 diabetes dataset identifies a list of protein candidates that is in very good agreement with previously performed transcriptomics meta analysis. Making use of quantitative properties of signal patterns identified, PPINGUIN can reveal new isoform candidates. Conclusions Regarding the increasing importance of quantitative proteomics we think that this method will be useful in practical applications like model fitting or functional enrichment analysis. We recommend to use this method if quantitation is a major objective of research.

  5. Comparison of Protein Expression Profiles of Different Stages of Lymph Nodes Metastasis in Breast Cancer

    Hui-Hua Lee, Chu-Ai Lim, Yew-Teik Cheong, Manjit Singh, Lay-Harn Gam

    2012-01-01

    Full Text Available Breast cancer is the most common cancer among women worldwide. Breast cancer metastasis primarily happens through lymphatic system, where the extent of lymph node metastasis is the major factor influencing staging, prognosis and therapeutic decision of the disease. We aimed to study the protein expression changes in different N (regional lymph nodes stages of breast cancer. Protein expression profiles of breast cancerous and adjacent normal tissues were mapped by proteomics approach that comprises of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and tandem mass spectrometry (LC-MS/MS analysis. Calreticulin and tropomyosin alpha 3 chains were the common up-regulated proteins in N0, N1 and N2 stages of breast cancer. Potential biomarker for each N stage was HSP 70 for N0, 80 k protein H precursor and PDI for N1 stage while 78 kDa glucose-regulated protein was found useful for N2 stage. In addition, significant up-regulation of PDI A3 was detected only in the metastasized breast cancer. The up-regulation expression of these proteins in cancerous tissues can potentially use as indicators for diagnosis, treatment and prognosis of different N stages of breast cancer.

  6. Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

  7. Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer

    Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E; Cold, Søren; Tan, Qihua; Depont Christensen, René; Mogensen, Ole; Kruse, Torben; Madsen, Jonna Skov; Jensen, Ole Nørregaard

    2008-01-01

    Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a...... of nine mass spectrometric protein profiles were obtained for each serum sample. A total of 533 common peaks were defined and represented a 'reference protein profile'. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between...... cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss...

  8. Protein profiling reveals consequences of lifestyle choices on predicted biological aging.

    Enroth, Stefan; Enroth, Sofia Bosdotter; Johansson, Åsa; Gyllensten, Ulf

    2015-01-01

    Ageing is linked to a number of changes in how the body and its organs function. On a molecular level, ageing is associated with a reduction of telomere length, changes in metabolic and gene-transcription profiles and an altered DNA-methylation pattern. Lifestyle factors such as smoking or stress can impact some of these molecular processes and thereby affect the ageing of an individual. Here we demonstrate by analysis of 77 plasma proteins in 976 individuals, that the abundance of circulating proteins accurately predicts chronological age, as well as anthropometrical measurements such as weight, height and hip circumference. The plasma protein profile can also be used to identify lifestyle factors that accelerate and decelerate ageing. We found smoking, high BMI and consumption of sugar-sweetened beverages to increase the predicted chronological age by 2-6 years, while consumption of fatty fish, drinking moderate amounts of coffee and exercising reduced the predicted age by approximately the same amount. This method can be applied to dried blood spots and may thus be useful in forensic medicine to provide basic anthropometrical measures for an individual based on a biological evidence sample. PMID:26619799

  9. Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

    Scicchitano, Marshall S.; Dalmas, Deidre A.; Boyce, Rogely W.; Thomas, Heath C.; Frazier, Kendall S.

    2009-01-01

    Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples ...

  10. Discovery of protein profiles for differentiated thyroid cancer using SELDI TOF MS

    Low sensitivity of diagnostic whole body iodine scintigraphy and intermediate range of serum thyroglobulin (Tg) with or without anti-Tg antibody make it difficult to select the patients with differentiated thyroid cancer who need further treatment. Surfaced Enhanced Laser Desorption /Ionization - Time of Flight - Mass Spectrometry (SELDI TOF MS) is a useful method to evaluate cancer proteome, biomarkers and patterns of biomarkers. In this preliminary study, we evaluated and developed protein profiles for the discrimination between patients with differentiated thyroid cancer and non-cancer controls using SELDI technology. Serum samples from 10 healthy controls and from 14 patients with papillary thyroid cancer before thyroidectomy were analyzed by SELDI MS. Multiple protein peaks detected were analyzed by the computer software to develop a classifier for separating cancer patients form controls. The classifier was then challenged to 24 serum samples to determine the validity and accuracy of the classification system. All patients with papillary thyroid cancer had no other concomitant cancer or thyroiditis. Their serum Tg concentration was 55.8 (1.5 - 249.7) and 2 patients had extra-thyroidal extension. According to the SELDI analysis, protein peaks at 3696 Da, 4178 Da, and 8149 Da were more prominent in cancer patients than controls in various degrees. Among those, protein peak at 4178 Da was determined as classifier by computer software, and the sensitivity, specificity and accuracy for discrimination of cancer patients from controls was 92.9% (13/14), 90% (9/10) and 91.7% respectively. This preliminary study suggests that serum protein profiles of differentiated thyroid cancer can be used for differentiation between cancer patients and non-cancer controls. And further clinical studies in various test sets will offer useful information in selecting patients who require treatment

  11. The Activity-Based Approach

    McNally, Michael G.; Rindt, Craig

    2008-01-01

    What is the activity-based approach (ABA) and how does it differ from the conventional trip-based model of travel behavior? From where has the activity approach evolved, what is its current status, and what are its potential applications in transportation forecasting and policy analysis. What have been the contributions of activity-based approaches to understanding travel behavior? The conventional trip-based model of travel demand forecasting (see Chapters 2 and 3) has always lacked...

  12. Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

    J Vejayan

    2010-01-01

    Full Text Available Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI and molecular weight (MW. Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family - Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma - were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa. Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.

  13. Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage.

    Sakai, Yosuke; Miyake, Ariko; Doi, Naoya; Sasada, Hikari; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution. PMID:27536295

  14. Hypothalamic protein profiles associated with inhibited feed intake of ducks fed with insufficient dietary arginine.

    Wang, C; Zheng, A J; Xie, M; Huang, W; Xie, J J; Hou, S S

    2014-07-01

    An experiment was conducted to investigate the effect of arginine on feed intake regulation. One hundred and twenty six 1-day-old male White Pekin ducks (Anas platyrhynchos domestica) were randomly were allotted to one of two dietary treatments. The birds were fed diets containing 0.71% (deficient) or 1.27% (sufficient) arginine for 3 weeks. At 21 days of age, feed intake was determined and hypothalamic protein profiles were analyzed using isobaric tags for relative and absolute quantification technique. The birds fed with arginine-deficient diet had a lower final live BW and cumulative feed intake (P1.5-fold expressional changes between arginine-deficient and -sufficient dietary treatments. Nine of these proteins were upregulated and seven of them were downregulated. The identified proteins could be regrouped into six categories: protein processing, carbohydrate metabolism and energy production, transporter, cytoskeleton, immunity and neuronal development. Dietary arginine deficiency decreased expression of proteins involved in energy production (glycine amidinotransferase, aldolase B fructose-bisphosphate, aconitase, transaldolase, 6-phosphofructokinase type C-like) and oxygen transportation (haemoglobin subunit α expression). The proteomic alterations described here provides valuable insights into the interactions of arginine with appetite. PMID:24804691

  15. Protein-ligand-based pharmacophores: generation and utility assessment in computational ligand profiling.

    Meslamani, Jamel; Li, Jiabo; Sutter, Jon; Stevens, Adrian; Bertrand, Hugues-Olivier; Rognan, Didier

    2012-04-23

    Ligand profiling is an emerging computational method for predicting the most likely targets of a bioactive compound and therefore anticipating adverse reactions, side effects and drug repurposing. A few encouraging successes have already been reported using ligand 2-D similarity searches and protein-ligand docking. The current study describes the use of receptor-ligand-derived pharmacophore searches as a tool to link ligands to putative targets. A database of 68,056 pharmacophores was first derived from 8,166 high-resolution protein-ligand complexes. In order to limit the number of queries, a maximum of 10 pharmacophores was generated for each complex according to their predicted selectivity. Pharmacophore search was compared to ligand-centric (2-D and 3-D similarity searches) and docking methods in profiling a set of 157 diverse ligands against a panel of 2,556 unique targets of known X-ray structure. As expected, ligand-based methods outperformed, in most of the cases, structure-based approaches in ranking the true targets among the top 1% scoring entries. However, we could identify ligands for which only a single method was successful. Receptor-ligand-based pharmacophore search is notably a fast and reliable alternative to docking when few ligand information is available for some targets. Overall, the present study suggests that a workflow using the best profiling method according to the protein-ligand context is the best strategy to follow. We notably present concrete guidelines for selecting the optimal computational method according to simple ligand and binding site properties. PMID:22480372

  16. Storage protein profiles in Spanish and runner market type peanuts and potential markers

    Luo M

    2006-10-01

    Full Text Available Abstract Background Proteomic analysis has proven to be the most powerful method for describing plant species and lines, and for identification of proteins in complex mixtures. The strength of this method resides in high resolving power of two-dimensional electrophoresis (2-DE, coupled with highly sensitive mass spectrometry (MS, and sequence homology search. By using this method, we might find polymorphic markers to differentiate peanut subspecies. Results Total proteins extracted from seeds of 12 different genotypes of cultivated peanut (Arachis hypogaea L., comprised of runner market (A. hypogaea ssp. hypogaea and Spanish-bunch market type (A. hypogaea ssp. fastigiata, were separated by electrophoresis on both one- and two-dimensional SDS-PAGE gels. The protein profiles were similar on one-dimensional gels for all tested peanut genotypes. However, peanut genotype A13 lacked one major band with a molecular weight of about 35 kDa. There was one minor band with a molecular weight of 27 kDa that was present in all runner peanut genotypes and the Spanish-derivatives (GT-YY7, GT-YY20, and GT-YY79. The Spanish-derivatives have a runner-type peanut in their pedigrees. The 35 kDa protein in A13 and the 27 kDa protein in runner-type peanut genotypes were confirmed on the 2-D SDS-PAGE gels. Among more than 150 main protein spots on the 2-D gels, four protein spots that were individually marked as spots 1–4 showed polymorphic patterns between runner-type and Spanish-bunch peanuts. Spot 1 (ca. 22.5 kDa, pI 3.9 and spot 2 (ca. 23.5 kDa, pI 5.7 were observed in all Spanish-bunch genotypes, but were not found in runner types. In contrast, spot 3 (ca. 23 kDa, pI 6.6 and spot 4 (ca. 22 kDa, pI 6.8 were present in all runner peanut genotypes but not in Spanish-bunch genotypes. These four protein spots were sequenced. Based on the internal and N-terminal amino acid sequences, these proteins are isoforms (iso-Ara h3 of each other, are iso-allergens and may be

  17. Analysis of whey protein hydrolysates: peptide profile and ACE inhibitory activity

    Marialice Pinto Coelho Silvestre; Mauro Ramalho Silva; Viviane Dias Medeiros Silva; Mariana Wanessa Santana de Souza; Carlos de Oliveira Lopes Junior; Wendel de Oliveira Afonso

    2012-01-01

    The aim of this study was to prepare enzymatic hydrolysates from whey protein concentrate with a nutritionally adequate peptide profile and the ability to inhibit angiotensin-converting enzyme (ACE) activity. The effects of the type of enzyme used (pancreatin or papain), the enzyme:substrate ratio (E:S ratio=0.5:100, 1:100, 2:100 and 3:100) and the use of ultrafiltration (UF) were investigated. The fractionation of peptides was performed by size-exclusion-HPLC, and the quantification of the c...

  18. Seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in Brahman bulls.

    Rego, J P A; Moura, A A; Nouwens, A S; McGowan, M R; Boe-Hansen, G B

    2015-09-01

    The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively. PMID:26282524

  19. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.

    van den Brink-van der Laan, Els; Killian, J Antoinette; de Kruijff, Ben

    2004-11-01

    Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure. PMID:15519321

  20. Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer

    Becker, Marc A.; Ibrahim, Yasir H.; Oh, Annabell S.; Fagan, Dedra H.; Byron, Sara A.; Sarver, Aaron L.; Lee, Adrian V.; Shaw, Leslie M.; Fan, Cheng; Perou, Charles M.; Yee, Douglas

    2016-01-01

    Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer. PMID:26991655

  1. Serum protein profile study of clinical samples using high performance liquid chromatography-laser induced fluorescence: case of cervical and oral cancers

    Karemore, Gopal; Sujatha, .; Rai, Lavanya; Pai, Keerthilatha M.; Kartha, V. B.; Santhosh C., .

    2009-02-01

    The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for establishing the ability of HPLC-LIF protein profiling technique for discrimination, using hard clustering and Fuzzy clustering methods. The clustering algorithms have quite successfully classified the profiles as belonging to normal, cancer of cervix, and oral cancer conditions.

  2. Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

    2009-01-01

    During the last decade,increasing evidence suggested that bone marrow stromal cells(MSCs) have the potential to differentiate into neural lineages.Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions.However,no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported.In this study,we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions.By using two-dimensional gel electrophoresis(2-DE),we compared the protein profiles of MSCs before and after induced differentiation.We obtained 792 protein spots in the protein profile by 2-DE,and found that 74 spots changed significantly before and after the differentiation using PDQuest software,with 43 up-regulated and 31 down-regulated.We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS) and by database searching,and found that they could be grouped into various classes,including cytoskeleton and structure proteins,growth factors,metabolic proteins,chaperone proteins,receptor proteins,cell cycle proteins,calcium binding proteins,and other proteins.These proteins also include neural and glial proteins,such as BDNF,CNTF and GFAP.The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

  3. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I.-Ping; Camargo, Eddie; Tzen, Jason T. C.; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-10-01

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  4. Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.

    Chong Zhu

    Full Text Available Protein disulfide isomerases (PDI are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2 contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2 induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the

  5. Protein profiling and histone deacetylation activities in somaclonal variants of oil palm (Elaeis guineensis Jacq.).

    Yaacob, Jamilah Syafawati; Loh, Hwei-San; Mat Taha, Rosna

    2013-01-01

    Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs), involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets) and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants. PMID:23844406

  6. Protein Profiling and Histone Deacetylation Activities in Somaclonal Variants of Oil Palm (Elaeis guineensis Jacq.

    Jamilah Syafawati Yaacob

    2013-01-01

    Full Text Available Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs, involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants.

  7. Micropropagation and protein profile analysis by SDS-PAGE of Gracilaria changii (Rhodophyta, Solieriaceae

    Lin Wei Jong

    2015-05-01

    Full Text Available Gracilaria changii seaweed is primarily important as a source of agar with wide applications in food industries. The high demand of agar led to gradual depletion of G. changii in natural resources. Establishment of in vitro culture of G. changii has an important role and allowing G. changii explants to grow optimally under controlled conditions to provide constant, continuous and sufficient seedlings supply for Gracilaria farming. This study focused on micropropagation culture of G. changii in which different exogenous factors influencing seaweed growth were investigated: strength of chosen medium Provasoli’s enriched seawater (PES, types and concentration of fertilizers/biostimulant, supplementation of plant growth regulators and seawater salinity. The results were presented in daily growth rate of explants and data analysis was carried out using one-way ANOVA. The results demonstrated high growth rate of G. changii in 25% of PES supplemented with 5 mg L−1 AMPEP, and seawater salinity range between 30 and 40 ppt, respectively. Protein profiles of tissue-cultured and farm cultivated G. changii were produced by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. The results demonstrated no remarkable difference in the protein profiles and indicated the suitability of the culture condition for the growth of G. changii.

  8. Methionine sulfoxide profiling of milk proteins to assess the influence of lipids on protein oxidation in milk.

    Wüst, Johannes; Pischetsrieder, Monika

    2016-06-15

    Thermal treatment of milk and milk products leads to protein oxidation, mainly the formation of methionine sulfoxide. Reactive oxygen species, responsible for the oxidation, can be generated by Maillard reaction, autoxidation of sugars, or lipid peroxidation. The present study investigated the influence of milk fat on methionine oxidation in milk. For this purpose, quantitative methionine sulfoxide profiling of all ten methionine residues of β-lactoglobulin, α-lactalbumin, and αs1-casein was carried out by ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry with scheduled multiple reaction monitoring (UHPLC-ESI-MS/MS-sMRM). Analysis of defatted and regular raw milk samples after heating for up to 8 min at 120 °C and analysis of ultrahigh-temperature milk samples with 0.1%, 1.5%, and 3.5% fat revealed that methionine oxidation of the five residues of the whey proteins and of residues M 123, M 135, and M 196 of αs1-casein was not affected or even suppressed in the presence of milk fat. Only the oxidation of residues M 54 and M 60 of αs1-casein was promoted by lipids. In evaporated milk samples, formation of methionine sulfoxide was hardly influenced by the fat content of the samples. Thus, it can be concluded that lipid oxidation products are not the major cause of methionine oxidation in milk. PMID:26927981

  9. Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

    Yu, Tao; Tao, Yonghui; Yang, Meiqiang; Chen, Peng; Gao, XiaoBo; Zhang, Yanbo; Zhang,Tao; Chen, Zi; Hou, Jian; Zhang, Yan; Ruan, Kangcheng; Wang, Hongyan; Hu, Ronggui

    2014-01-01

    Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be a...

  10. ACUTE PHASE PROTEINS, LIPID PROFILE AND PROINFLAMMATORY CYTOKINES IN HEALTHY AND BRONCHOPNEUMONIC WATER BUFFALO CALVES

    Sabry M. El-Bahr

    2013-01-01

    Full Text Available The aim of the present study was to evaluate the diagnostic value of Acute Phase Proteins (APP, lipid profiles and proinflammatory cytokines in healthy and bronchopneumonic water buffalo calves. Therefore, sixty water buffalo calves (9±1 month old, 175±15 kg were divided into two equal groups, the first group represented healthy, control, calves whereas calves of the second group were affected with bronchopneumonia. Total leukocytic and differential counts were determined. Serum total protein, albumin, Triacylglyceol (TAG, low density lipoprotein cholesterol (LDL-c, High Density Lipoprotein cholesterol (HDL-c, Total cholesterol, Alanine Amino Transferase (ALT, Aspartate Amino Transferase (AST, Alkaline Phosphatase (ALP, Fibrinogen (Fb, Haptaglobin (Hp, Serum Amyloid A (SAA, Tumor Necrosis Factor-alpha (TNF-α, Interleukins (IL1β, IL-12 and Interferon-gamma (IFN-γ were also determined. In addition, Bronchoalveolar Lavage (BAL was collected and analyzed. The present findings indicated that, total leukocytic and neutrophils counts were significantly (p<0.05 higher in pneumonic water buffalo calves compare with control. The examined biochemical parameters were significantly (p<0.05 increased in pneumonic calves except for total protein, albumin, cholesterol and HDL-c which were significantly (p<0.05 lower compare with control. Serum concentrations of investigated APP and proinflammatory cytokines were significantly (p<0.05 higher in pneumonic water buffalo calves than those of control. The present study demonstrated that, APP, lipid profile and proinflammatory cytokines perhaps served as biomarkers of bronchopneumonia in water buffalo calves. However, future studies with higher baseline sampling are still needed to establish and validate reference values for APP and cytokines in water buffalo calves.

  11. Maternal serum protein profile and immune response protein subunits as markers for non-invasive prenatal diagnosis of trisomy 21, 18, and 13

    Narasimhan, Kothandaraman

    2013-02-01

    Objectives: To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. Methods: We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. Results: Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. Conclusions: Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies. © 2013 John Wiley & Sons, Ltd.

  12. A comparative protein profile of mammalian erythrocyte membranes identified by mass spectrometry.

    Sharma, Savita; Punjabi, Vinny; Zingde, Surekha M; Gokhale, Sadashiv M

    2014-11-01

    A comparative analysis of erythrocyte membrane proteins of economically important animals, goat (Capra aegagrus hircus), buffalo (Bubalus bubalis), pig (Sus scrofa), cow (Bos tauras), and human (Homo sapiens) was performed. Solubilized erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE), visualized by staining the gels with Commassie Brilliant Blue (CBB), and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Emerging results show that all major erythrocyte membrane proteins present in human are also seen in all the animals except for band 4.5 which could not be identified. Band 3 is seen as more intense and compact, band 4.1 appears as a doublet in all the animal erythrocyte membranes, band 4.2 exhibits a slightly higher molecular weight (Mr) in buffalo, and cow and band 4.9 has a higher Mr in all the animals relative to the human protein. In addition, there are two new bands in the goat membrane, band G1, identified as HSP 90α, and band G2 identified as HSP 70. A new band C2 identified as HSP 70 is also seen in cow membranes. Peroxiredoxin II is of lower intensity and/or higher Mr in the animals. The difference in size of the proteins possibly indicates the variations in the composition of the amino acids. The difference in intensity of the proteins among these mammalians highlights the presence of less or more number of copies of that protein per cell. This data complement the earlier observations of differences in the sialoglycoprotein profile and effect of proteases and neuraminidase on agglutination among the mammalian erythrocytes. This study provides a platform to understand the molecular architecture of the individual erythrocytes, and in turn the dependent disorders, their phylogenetic relationship and also generates a database of erythrocyte membrane proteins of mammals. The animals selected for this study are of economic importance as

  13. Plasma protein profiling of mild cognitive impairment and Alzheimer's disease across two independent cohorts.

    Muenchhoff, Julia; Poljak, Anne; Song, Fei; Raftery, Mark; Brodaty, Henry; Duncan, Mark; McEvoy, Mark; Attia, John; Schofield, Peter W; Sachdev, Perminder S

    2015-01-01

    To unlock the full potential of disease modifying treatments, it is essential to develop early biomarkers for Alzheimer's disease (AD). For practical reasons, blood-based markers that could provide a signal at the stage of mild cognitive impairment (MCI) or even earlier would be ideal. Using the proteomic approach of isobaric tagging for relative and absolute quantitation (iTRAQ), we compared the plasma protein profiles of MCI, AD, and cognitively normal control subjects from two independent cohorts: the Sydney Memory and Ageing Study (261 MCI subjects, 24 AD subjects, 411 controls) and the Hunter Community Study (180 MCI subjects, 153 controls). The objective was to identify any proteins that are differentially abundant in MCI and AD plasma in both cohorts, since they might be of interest as potential biomarkers, or could help direct future mechanistic studies. Proteins representative of biological processes relevant to AD pathology, such as the complement system, the coagulation cascade, lipid metabolism, and metal and vitamin D and E transport, were found to differ in abundance in MCI. In particular, levels of complement regulators C1 inhibitor and factor H, fibronectin, ceruloplasmin, and vitamin D-binding protein were significantly decreased in MCI participants from both cohorts. Several apolipoproteins, including apolipoprotein AIV, B-100, and H were also significantly decreased in MCI. Most of these proteins have previously been reported as potential biomarkers for AD; however, we show for the first time that a significant decrease in plasma levels of two potential biomarkers (fibronectin and C1 inhibitor) is evident at the MCI stage. PMID:25159666

  14. Activity based costing (ABC Method

    Prof. Ph.D. Saveta Tudorache

    2008-05-01

    Full Text Available In the present paper the need and advantages are presented of using the Activity BasedCosting method, need arising from the need of solving the information pertinence issue. This issue has occurreddue to the limitation of classic methods in this field, limitation also reflected by the disadvantages ofsuch classic methods in establishing complete costs.

  15. Matrix metalloproteinase protein expression profiles cannot distinguish between normal and early osteoarthritic synovial fluid

    Heard Bryan J

    2012-07-01

    Full Text Available Abstract Background Osteoarthritis (OA and Rheumatoid arthritis (RA are diseases which result in the degeneration of the joint surface articular cartilage. Matrix Metalloproteinases (MMPs are enzymes that aid in the natural remodelling of tissues throughout the body including cartilage. However, some MMPs have been implicated in the progression of OA and RA as their expression levels and activation states can change dramatically with the onset of disease. Yet, it remains unknown if normal and arthritic joints demonstrate unique MMPs expression profiles, and if so, can the MMP expression profile be used to identify patients with early OA. In this study, the synovial fluid protein expression levels for MMPs 1, 2, 3, 7, 8, 9, 12 & 13, as well as those for the Tissue Inhibitors of MMPs (TIMPs 1, 2, 3, & 4 were examined in highly characterized normal knee joints, and knee joints with clinically diagnosed OA (early and advanced or RA. The purpose of this study was to determine if normal, OA, and RA patients exhibit unique expression profiles for a sub-set of MMPs, and if early OA patients have a unique MMP expression profile that could be used as an early diagnostic marker. Methods Synovial fluid was aspirated from stringently characterized normal knee joints, and in joints diagnosed with either OA (early and advanced or RA. Multiplexing technology was employed to quantify protein expression levels for 8 MMPs and 4 TIMPs in the synovial fluid of 12 patients with early OA, 17 patients diagnosed with advanced OA, 15 with RA and 25 normal knee joints. Principle component analysis (PCA was used to reveal which MMPs were most influential in the distinction between treatment groups. K – means clustering was used to verify the visual grouping of subjects via PCA. Results Significant differences in the expression levels of MMPs and TIMPs were observed between normal and arthritic synovial fluids (with the exception of MMP 12. PCA demonstrated that MMPs 2, 8

  16. In silico comparative analysis and expression profile of antioxidant proteins in plants.

    Sheoran, S; Pandey, B; Sharma, P; Narwal, S; Singh, R; Sharma, I; Chatrath, R

    2013-01-01

    The antioxidant system in plants is a very important defensive mechanism to overcome stress conditions. We examined the expression profile of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) using a bioinformatics approach. We explored secondary structure prediction and made detailed studies of signature pattern of antioxidant proteins in four plant species (Triticum aestivum, Arabidopsis thaliana, Oryza sativa, and Brassica juncea). Fingerprinting analysis was done with ScanProsite, which includes a large collection of biologically meaningful signatures. Multiple sequence alignment of antioxidant proteins of the different plant species revealed a conserved secondary structure region, indicating homology at the sequence and structural levels. The secondary structure prediction showed that these proteins have maximum tendency for α helical structure. The sequence level similarities were also analyzed with a phylogenetic tree using neighbor-joining method. In the antioxidant enzymes SOD, CAT and APX, three major families of signature were predominant and common; these were PKC_PHOSPHO_SITE, CK2_PHOSPHO_SITE and N-myristoylation site, which are functionally related to various plant signaling pathways. This study provides new strategies for screening of biomodulators involved in plant stress metabolism that will be useful for designing degenerate primers or probes specific for antioxidant. These enzymes could be the first line of defence in the cellular antioxidant defence pathway, activated due to exposure to abiotic stresses. PMID:23512671

  17. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Muhammad Ibrahim

    Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  18. Changes in the serum protein profile during radiotherapy to the upper respiratory and gastro-intestinal tracts

    Patients with a cancer of the upper airways of upper gastro-intestinal tract present a state of malnutrition as a result of the disease itself and, more importantly, as a result of its localisation. Loco-regional radiotherapy often leads to an aggravation, of this state. The protein profile, consisting of nine serum proteins, was determined each week in 54 patients with cancer of the upper respirato-gastro-intestinal tract receiving radiotherapy. During the course of radiotherapy, the already altered nutritional state of these patients deteriorated further, as shown by a regular and significant downturn in the weight curve. The weekly monitoring of the protein profile showed a gradual and significant decrease in the levels of nutritional proteins (prealbumin, retinol binding protein, transferrin) and immunoglobulins (IgM, IgA) and a small variation in the levels of inflammatory proteins (haptoglobin, orosomucoid, C3 complement fraction, alpha1-antitrypsin). The protein profile, established on the basis of carefully selected proteins, can provide useful information in the monitoring of a patient's nutritional state

  19. PENGARUH KOAGULAN DAN KONDISI KOAGULASI TERHADAP PROFIL PROTEIN CURD KEDELAI SERTA KORELASINYA TERHADAP TEKSTUR [Effect of Coagulant and Coagulation Condition to Soybean Curd Protein Profile and Its Correlation to Texture

    Dahrul Syah1,2*

    2012-06-01

    Full Text Available The research aims to study the effect of coagulation parameters to the electrophoretical profile of coagulated soybean protein as well as to texture properties and perception. Several coagulation parameters such as type of coagulant, concentration or age of coagulant, and coagulation temperature were studied. The type of coagulant used in this study were GDL (glucono delta Lactone and tofu whey. The concentrations of GDL were 0.4%, 0.8%, and 1.2% and the ages of tofu whey were 1 day, 2 days, and 3 days. Two coagulation temperatures, 60°C and 80°C, were applied. The results of curd protein profile and texture profile analysis showed that coagulant type and coagulation condition did not affect the electrophoretical profile of soybean coagulated protein. However, the proportion of each subunit was affected significantly. Texture profiles, such as hardness, cohesiveness, and gumminess, were also affected by coagulant type and coagulation condition. Based on the subunits proportion, it can be concluded that 11 S and 7S protein proportions as well as the 11S/7S ratio correlated significantly with the hardness and gumminess of soybean curd made by tofu whey.

  20. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

  1. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Laura Camacho

    Full Text Available Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM versus non-brain metastatic (non-BM cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210 and two down-regulated miRNAs (miR-19a and miR-29c in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  2. Altered Plasma Profile of Antioxidant Proteins as an Early Correlate of Pancreatic β Cell Dysfunction.

    Kuo, Taiyi; Kim-Muller, Ja Young; McGraw, Timothy E; Accili, Domenico

    2016-04-29

    Insulin resistance and β cell dysfunction contribute to the pathogenesis of type 2 diabetes. Unlike insulin resistance, β cell dysfunction remains difficult to predict and monitor, because of the inaccessibility of the endocrine pancreas, the integrated relationship with insulin sensitivity, and the paracrine effects of incretins. The goal of our study was to survey the plasma response to a metabolic challenge in order to identify factors predictive of β cell dysfunction. To this end, we combined (i) the power of unbiased iTRAQ (isobaric tag for relative and absolute quantification) mass spectrometry with (ii) direct sampling of the portal vein following an intravenous glucose/arginine challenge (IVGATT) in (iii) mice with a genetic β cell defect. By so doing, we excluded the effects of peripheral insulin sensitivity as well as those of incretins on β cells, and focused on the first phase of insulin secretion to capture the early pathophysiology of β cell dysfunction. We compared plasma protein profiles with ex vivo islet secretome and transcriptome analyses. We detected changes to 418 plasma proteins in vivo, and detected changes to 262 proteins ex vivo The impairment of insulin secretion was associated with greater overall changes in the plasma response to IVGATT, possibly reflecting metabolic instability. Reduced levels of proteins regulating redox state and neuronal stress markers, as well as increased levels of coagulation factors, antedated the loss of insulin secretion in diabetic mice. These results suggest that a reduced complement of antioxidants in response to a mixed secretagogue challenge is an early correlate of future β cell failure. PMID:26917725

  3. Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

    Zehr Emilie S; Lavrov Dennis V; Tabatabai Louisa B

    2012-01-01

    Abstract Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for dis...

  4. Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris.

    Kappos, T; John, M A; Hussain, Z; Valvano, M A

    1992-01-01

    Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and th...

  5. Differential expression profiling of serum proteins and metabolites for biomarker discovery

    Roy, Sushmita Mimi; Anderle, Markus; Lin, Hua; Becker, Christopher H.

    2004-11-01

    A liquid chromatography-mass spectrometry (LC-MS) proteomics and metabolomics platform is presented for quantitative differential expression analysis. Proteome profiles obtained from 1.5 [mu]L of human serum show ~5000 de-isotoped and quantifiable molecular ions. Approximately 1500 metabolites are observed from 100 [mu]L of serum. Quantification is based on reproducible sample preparation and linear signal intensity as a function of concentration. The platform is validated using human serum, but is generally applicable to all biological fluids and tissues. The median coefficient of variation (CV) for ~5000 proteomic and ~1500 metabolomic molecular ions is approximately 25%. For the case of C-reactive protein, results agree with quantification by immunoassay. The independent contributions of two sources of variance, namely sample preparation and LC-MS analysis, are respectively quantified as 20.4 and 15.1% for the proteome, and 19.5 and 13.5% for the metabolome, for median CV values. Furthermore, biological diversity for ~20 healthy individuals is estimated by measuring the variance of ~6500 proteomic and metabolomic molecular ions in sera for each sample; the median CV is 22.3% for the proteome and 16.7% for the metabolome. Finally, quantitative differential expression profiling is applied to a clinical study comparing healthy individuals and rheumatoid arthritis (RA) patients.

  6. Gene expression profile of amyloid beta protein-injected mouse model for Alzheimer disease

    Ling-na KONG; Ping-ping ZUO; Liang MU; Yan-yong LIU; Nan YANG

    2005-01-01

    Aim: To investigate the gene expression profile changes in the cerebral cortex of mice injected icv with amyloid beta-protein (Aβ) fragment 25-35 using cDNA microarray. Methods: Balb/c mice were randomly divided into a control group and Aβ-treated group. The Morris water maze test was performed to detect the effect of Aβ-injection on the learning and memory of mice. Atlas Mouse 1.2 Expression Arrays containing 1176 genes were used to investigate the gene expression pattern of each group. Results: The gene expression profiles showed that 19 genes including TBX1, NF-κB, AP-1/c-Jun, cadherin, integrin, erb-B2, and FGFR1 were up-regulated after 2 weeks oficv administration of Aβ; while 12 genes were downregulated, including NGF, glucose phosphate isomerase 1, AT motif binding factor 1, Na+/K+-ATPase, and Akt. Conclusions: The results provide important leads for pursuing a more complete understanding of the molecular events of Aβ-injection into mice with Alzheimer disease.

  7. Gene expression profiles of novel caprine placental prolactin-related proteins similar to bovine placental prolactin-related proteins

    Sato Eimei

    2007-03-01

    Full Text Available Abstract Background This study reports the identification of a full-length cDNA sequence for two novel caprine prolactin-related proteins (cPRP1 and cPRP6, and their localization and quantitative expression in the placenta. Caprine PRPs are compared with known bovine PRPs. We examined their evolution and role in the ruminant placenta. Results Full-length cPRP1 and cPRP6 cDNA were cloned with a 717- and 720- nucleotide open-reading frame corresponding to proteins of 238 and 239 amino acids. The cPRP1 predicted amino acid sequence shares a 72% homology with bovine PRP1 (bPRP1. The cPRP6 predicted amino acid sequence shares a 74% homology with bovine PRP6 (bPRP6. The two cPRPs as well as bPRPs were detected only in the placentome by RT-PCR. Analysis by in situ hybridization revealed the presence of both cPRPs mRNA in the trophoblast binucleate cells. These mRNA were quantified by real-time RT-PCR analysis of the placentome at 30, 50, 90 and 140 days of pregnancy. Both new cPRP genes were able to translate a mature protein in a mammalian cell-expression system. Western blotting established the molecular sizes of 33 kDa for cPRP1 with FLAG-tag and 45 kDa for cPRP6 with FLAG-tag. The sequence properties and localized expression of cPRP1 and cPRP6 were similar to those of bovine. However, their expression profiles differed from those in bovine placenta. Although this study demonstrated possible roles of PRPs in caprine placenta, PRPs may regulate binucleate-cell functions like those in bovine, but their crucial roles are still unclear. Conclusion We have identified the novel PRPs in caprine placenta. Localization and quantitative expression of caprine PRPs were compared with bovine PRPs. The data indicate that PRP genes in caprine placenta have coordination functions for gestation, as they do in bovine. This is the first study of PRPs function in caprine placenta.

  8. Activity-Based Probe for N-Acylethanolamine Acid Amidase.

    Romeo, Elisa; Ponzano, Stefano; Armirotti, Andrea; Summa, Maria; Bertozzi, Fabio; Garau, Gianpiero; Bandiera, Tiziano; Piomelli, Daniele

    2015-09-18

    N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme. PMID:26102511

  9. MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles

    Reinert, Line; Shi, B; Nandi, S; Mazan-Mamczarz, K; Vitolo, M; Bachman, KE; He, H; Gartenhaus, RB

    2006-01-01

    MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification...... enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up......-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis....

  10. Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria.

    Min, Fangui; Pan, Jinchun; Wu, Ruike; Chen, Meiling; Kuang, Huiwen; Zhao, Weibo

    2016-02-14

    Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection. PMID:26437786

  11. Apoptotic protein profile in Leishmania donovani after treatment with hexaazatrinaphthylenes derivatives.

    López-Arencibia, Atteneri; Martín-Navarro, Carmen M; Sifaoui, Ines; Reyes-Batlle, María; Wagner, Carolina; Lorenzo-Morales, Jacob; Piñero, José E

    2016-07-01

    Two hexaazatrinaphthylene derivatives, DGV-B and DGV-C previously known to induce an apoptotic-like process in Leishmania donovani parasites were used in this study. For this purpose, two different human protein commercial arrays were used to determine the proteomic profile of the treated parasites compared to non-treated ones. One of the commercial arrays is able to detect the relative expression of 35 human apoptosis-related proteins and the other one is able to identify 9 different human kinases. The obtained results showed that the two tested molecules were able to activate a programmed cell death process by different pathways in the promastigote stage of the parasite. The present study reports the potential application of two commercialised human apoptotic arrays to evaluate the action mechanism of active compounds at least against Leishmania donovani. The obtained data would be useful to establish the putative activated apoptosis pathways in the treated parasites and to further support the use of hexaazatrinaphthylene derivatives for the treatment of leishmaniasis in the near future. Nevertheless, further molecular studies should be developed in order to design and evaluate specific apoptotic arrays for Leishmania genus. PMID:27060614

  12. Rapid protein structure classification using one-dimensional structure profiles on the bioSCAN parallel computer.

    Hoffman, D L; Laiter, S; Singh, R K; Vaisman, I I; Tropsha, A

    1995-12-01

    Rapid growth of protein structures database in recent years requires an effective approach for objective comparison and classification of deposited protein structures. We describe a novel method for structure comparison and classification based on the alignment of one-dimensional structure profiles. These profiles are obtained by calculating the OCCO pseudodihedral angles (formed by O-C-C-O atoms of carbonyl groups of consecutive amino acid residues) from protein three-dimensional coordinates. These angle measurements are then converted into a 24 letter alphabet, and the protein structures are represented by sequences of letter from this alphabet. The BioSCAN parallel computer, designed for primary sequence alignment, is used to rapidly align and classify these one-dimensional structure profiles. We have developed and implemented weighted scoring matrix to identify structural classes based on commonly found structural motifs. The results of our experiments are in good agreement with the traditional protein structure classification schemes. One-dimensional structure profiles significantly improve efficiency of structure comparison and classification. PMID:8808584

  13. Effect of whey protein on blood lipid profiles: a meta-analysis of randomized controlled trials.

    Zhang, J-W; Tong, X; Wan, Z; Wang, Y; Qin, L-Q; Szeto, I M Y

    2016-08-01

    Previous studies have suggested that whey supplementation may have beneficial effects on lipid profiles, although results were inconsistent. A literature search was performed in March 2015 for randomized controlled trials observing the effects of whey protein and its derivatives on circulating levels of triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). A meta-analysis was subsequently conducted. The meta-analysis results of 13 trials showed that whey supplementation significantly reduced the circulating TG level by 0.11 mmol/l (95% CI: -0.21, 0 mmol/l), whereas the whey protein had no effects on circulating TC (-0.11 mmol/l, 95% CI: -0.27, 0.05 mmol/l), LDL-C (-0.08 mmol/l, 95% CI: -0.23, 0.07 mmol/l) and HDL-C (0.01 mmol/l, 95% CI: -0.04, 0.05 mmol/l). Subgroup analysis showed that significant TG reduction disappeared in participants with low body mass index, low supplemental whey dose or under exercise training/energy restriction during the trial. No evidence of heterogeneity across studies and publication bias was observed. In conclusion, our findings demonstrated that the effects of whey protein supplementation were modest, with an overall lowering effect on TG but no effect on TC, LDL-C and HDL-C. PMID:27026427

  14. Expression profiles of hydrophobic surfactant proteins in children with diffuse chronic lung disease

    Guttentag Susan

    2005-07-01

    Full Text Available Abstract Background Abnormalities of the intracellular metabolism of the hydrophobic surfactant proteins SP-B and SP-C and their precursors may be causally linked to chronic childhood diffuse lung diseases. The profile of these proteins in the alveolar space is unknown in such subjects. Methods We analyzed bronchoalveolar lavage fluid by Western blotting for SP-B, SP-C and their proforms in children with pulmonary alveolar proteinosis (PAP, n = 15, children with no SP-B (n = 6, children with chronic respiratory distress of unknown cause (cRD, n = 7, in comparison to children without lung disease (n = 15 or chronic obstructive bronchitis (n = 19. Results Pro-SP-B of 25–26 kD was commonly abundant in all groups of subjects, suggesting that their presence is not of diagnostic value for processing defects. In contrast, pro-SP-B peptides cleaved off during intracellular processing of SP-B and smaller than 19–21 kD, were exclusively found in PAP and cRD. In 4 of 6 children with no SP-B, mutations of SFTPB or SPTPC genes were found. Pro-SP-C forms were identified at very low frequency. Their presence was clearly, but not exclusively associated with mutations of the SFTPB and SPTPC genes, impeding their usage as candidates for diagnostic screening. Conclusion Immuno-analysis of the hydrophobic surfactant proteins and their precursor forms in bronchoalveolar lavage is minimally invasive and can give valuable clues for the involvement of processing abnormalities in pediatric pulmonary disorders.

  15. Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

    Kuballa Anna V

    2007-10-01

    Full Text Available Abstract Background Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992 previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path

  16. Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles

    Dobrovolskaia, Marina A.; Patri, Anil K.; Zheng, Jiwen; Clogston, Jeffrey D.; Ayub, Nader; Aggarwal, Parag; Neun, Barry W.; Hall, Jennifer B.; McNeil, Scott E.

    2008-01-01

    Nanoparticle size and plasma binding profile contribute to a particle’s longevity in the bloodstream, which can have important consequences for therapeutic efficacy. In this study an approximate doubling in nanoparticle hydrodynamic size was observed upon in vitro incubation of 30- and 50-nm colloidal gold in human plasma. Plasma proteins that bind the surface of citrate-stabilized gold colloids have been identified. Effects of protein binding on the nanoparticle hydrodynamic size, elements o...

  17. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  18. Differential appearance of isoforms and cultivar variation in protein temporal profiles revealed in the maturing barley grain proteome

    Finnie, Christine; Bak-Jensen, K.S.; Laugesen, Sabrina; Roepstorff, P.; Svensson, Birte

    2006-01-01

    Proteome analysis of mature barley (Hordeum vulgare subsp. vulgare) seeds has led to the identification of proteins in about 450 spots on 2D-gels. To shed light on the role of some of these proteins, their temporal appearance was monitored over 5 weeks during grain-filling and maturation of field......-grown barley. Appearance profiles are described for 105 proteins identified in 185 2D-gel spots in the overlapping pI ranges 4-7 and 6-11. Grouping of proteins according to appearance across functional categories revealed instances of differential regulation of protein forms. Thus, a single 1-cys...... modification or defence against fungal pathogens. Comparison of two cultivars, Barke and Morex, led to identification of protein spots appearing earlier in Morex than Barke, reflecting the faster maturation of Morex seeds....

  19. Seasonal variations in the amino acid profile and protein nutritional value of Saccharina latissima cultivated in a commercial IMTA system

    Silva Marinho, Goncalo; Holdt, Susan Løvstad; Angelidaki, Irini

    2015-01-01

    Seaweeds have potential for the provision of biomass for food and feed supplements. The demand is increasing especially for proteins as ingredients; however, the amino acid profile is essential for evaluation of the nutritional value of proteins. The year-round protein concentration and amino acid...... Denmark in 2013–2014. Overall, there was no significant difference for the tested parameters between the two sampling sites; however, seasonal variations were found. The protein concentration varied markedly reaching a maximum of 10.8 % dry weight (DW) in November and a minimum of 1.3 % DW in May 2013.......9 % (based on WHO/FAO/UNU requirements) was achieved in November 2013. The presence of epiphytes in July to November changed neither the amino acid content nor the EAA score. S. latissima is comparable with wheat as a protein ingredient for fish feed and appears to be a suitable protein/amino acid source for...

  20. PERUBAHAN KANDUNGAN ANTIOKSIDAN, POLIFENOL DAN PROFIL PROTEIN SELAMA PRA-PERKECAMBAHA PADA BIJI KAKAO

    Kiki Ulfaniah

    2014-02-01

    Full Text Available [ENGLISH] Cocoa proven as food source that rich in bioactive compounds, especially polyphenols that have a role as an antioxidant because it can stop the free radical reactions. As the change in lifestyle of the people and the development of technology, various ways have been made to improve the nutrition quality of the cocoa beans with pre-germination methods. This research aimed to study the effect of pre-germination methods that change the content of antioxidants, polyphenols and protein profiles of cocoa beans. The research was conducted in the Laboratory of Genetics and Plant Breeding, Faculty of Agriculture, University of Jember, held from January 28 until April 30, 2013. Metodology research used Sulawesi cocoa beans type 1 beans without skin and with the seed coat, which is done with a completely randomized design (CRD in the old pre-germination 0, 1, 2, 3, 4, 5 days and repeated 4 times. The results showed that pre-germination methods provide a very real effect on the polyphenol content and antioxidant cocoa beans without without and the seed coat. The highest antioxidant content of cocoa beans without the skin of 0.47 µg/mg at the old pre-germination for 3 days and cocoa beans with the seed coat by 0.41 µg/mg in the old pre-germination 5 days. Polyphenol content of cocoa beans without skin and with the best seed coat that is in the control treatment had the highest content of 2.67 µg/mg for cocoa beans without skin and 2.46 µg/mg for cocoa beans with the seed coat. Keywords: Cocoa; Polyphenols; Antioxidants; Free Radicals; Pre-Germination [INDONESIAN] Kakao terbukti sebagai sumber makanan kaya senyawa bioaktif terutama polifenol yang mempunyai kasiat sebagai antioksidan karena mampu menghentikan reaksi radikal bebas. Seiring perubahan pola hidup masyarakat dan berkembangnya teknologi, berbagai cara telah dilakukan untuk meningkatkan kualitas nutrisi pada biji kakao dengan metode pra-perkecambahan. Penelitian ini bertujuan untuk

  1. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

    Wei-ying ZHANG; Fu-qing XU; Chang-liang SHAN; Rong XIANG; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model.Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes, Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2).Results: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion.Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the car-cinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

  2. Effects of breed and casein genetic variants on protein profile in milk from Swedish Red, Danish Holstein, and Danish Jersey

    Gustavsson, Frida; Buitenhuis, Albert Johannes; Johansson, M; Bertelsen, Henriette Pasgaard; Glantz, M; Poulsen, Nina Aagaard; Månsson, H Lindmark; Stålhammer, Hans; Larsen, Lotte Bach; Bendixen, Christian; Paulsson, M; Andrén, A

    2014-01-01

    : Swedish Red (SR), Danish Holstein (DH), and Danish Jersey (DJ). The protein profile with relative concentrations of α-lactalbumin, β-lactoglobulin, and αS1-, αS2-, κ-, and β-CN was determined for each milk sample using capillary zone electrophoresis. The genetic variants of the αS1- (CSN1S1), β- (CSN2...

  3. P178-S Profiling the Phosphoproteome: Discovery of Treatment Dependent Protein Markers of p38 and MK2 Inhibition

    Strelitzer, T. J.; Warder, S. E.; Perron, D. C.; Kwak, S. S.; Kamens, J. S.; Tomlinson, M. J.; Rogers, J C

    2007-01-01

    Protein phosphorylation is a critical signal transduction event in many areas of therapeutic interest, including cancer and inflammation. Understanding of these signaling events, their role in disease, or their modulation by therapy, necessitates identification and characterization of phosphoproteins regulated by hormones, circulating factors, or pharmacological agents. Identification of regulated phosphoproteins requires sensitive and robust methods of phosphoprotein enrichment, profiling, i...

  4. Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss)

    Godiksen, Helene; Morzel, M.; Hyldig, Grethe; Jessen, Flemming

    2009-01-01

    Post-mortem softening of fish tissue often results in low yield and decreased product quality. In this study, proteolytic profiles of trout stored 5 days oil ice were obtained by SDS-PAGE. The link between protein hand intensities and firmness of trout fillets was examined through a correlation...

  5. Transcript Profiles of Two Wheat Lipid Transfer Protein-encoding Genes are Altered During Attach by Hessian Fly Larvae

    ‘GeneCalling’, an mRNA profiling technology, was used to identify a candidate lipid transfer protein (LTP) sequence that showed decreased mRNA abundance in wheat (Triticum aestivum L. em Thell) plants following attack by virulent Hessian fly (Mayetiola destructor Say) larvae (compatible interaction)...

  6. Analysis of whey protein hydrolysates: peptide profile and ACE inhibitory activity

    Marialice Pinto Coelho Silvestre

    2012-12-01

    Full Text Available The aim of this study was to prepare enzymatic hydrolysates from whey protein concentrate with a nutritionally adequate peptide profile and the ability to inhibit angiotensin-converting enzyme (ACE activity. The effects of the type of enzyme used (pancreatin or papain, the enzyme:substrate ratio (E:S ratio=0.5:100, 1:100, 2:100 and 3:100 and the use of ultrafiltration (UF were investigated. The fractionation of peptides was performed by size-exclusion-HPLC, and the quantification of the components of the chromatographic fractions was carried out by a rapid Corrected Fraction Area method. The ACE inhibitory activity (ACE-IA was determined by Reverse Phase-HPLC. All parameters tested affected both the peptide profile and the ACE-IA. The best peptide profile was achieved for the hydrolysates obtained with papain, whereas pancreatin was more advantageous in terms of ACE-IA. The beneficial effect of using a lower E:S ratio on the peptide profile and ACE-IA was observed for both enzymes depending on the conditions used to prepare the hydrolysates. The beneficial effect of not using UF on the peptide profile was observed in some cases for pancreatin and papain. However, the absence of UF yielded greater ACE-IA only when using papain.O objetivo deste estudo foi preparar hidrolisados enzimáticos do concentrado proteico do soro de leite com perfil peptídico nutricionalmente adequado e com capacidade para inibir a atividade da enzima conversora da angiotensina (ECA. Os efeitos do tipo de enzima usado (pancreatina ou papaína, da relação enzima:substrato (E:S=0,5:100, 1:100, 2:100 e 3:100 e do uso da ultrafiltração (UF foram investigados. O fracionamento dos peptídeos foi feito por CLAE de exclusão molecular e a quantificação dos componentes das frações cromatográficas foi realizada pelo método da Área Corrigida da Fração. A atividade inibitória da ECA (AI-ECA foi determinada por CLAE de fase reversa. Todos os parâmetros testados afetaram

  7. Protein content and electrophoretic profile of fat body and ovary extracts from workers of Melipona quadrifasciata anthidioides (Hymenoptera, Meliponini

    Vagner T. Paes de Oliveira

    Full Text Available Workers of Melipona quadrifasciata anthidioides (Lepeletier, 1836 develop their ovaries and lay eggs, therefore the production of vitellogenin is expected. In electrophoretic profiles only fat body extracts from nurse workers and ovary extracts from newly-emerged workers show protein with molecular mass similar to vitellogenin. However, an increase in the protein content was detected in forager fat body. This increase was attributed to storage of vitellogenin or other proteins in the previous phase and not discharged into the hemolymph or to an effect of the increased titre of juvenile hormone in this phase of worker life over the fat body functioning.

  8. The effect of thermal processing on protein quality and free amino acid profile of Terminalia catappa (Indian Almond) seed

    Adu, O. B.; Ogundeko, T. O.; Ogunrinola, O. O.; Saibu, G. M.; Elemo, B. O.

    2014-01-01

    The study examined the effect of various processing methods- boiling, drying and roasting- on the in vitro and in vivo protein digestibility and free amino acid profiles of Terminalia catappa seed. Moisture and crude protein of the various samples were determined. In vitro protein digestibility was determined after pepsin digestion. For the in vivo experiment, defatted T. catappa based diet was fed to 3 weeks old Wistar rats for 4 weeks and compared with animals maintained on casein based and...

  9. Suite of activity-based probes for cellulose-degrading enzymes.

    Chauvigné-Hines, Lacie M; Anderson, Lindsey N; Weaver, Holly M; Brown, Joseph N; Koech, Phillip K; Nicora, Carrie D; Hofstad, Beth A; Smith, Richard D; Wilkins, Michael J; Callister, Stephen J; Wright, Aaron T

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry and to increase enzyme active site inclusion for liquid chromatography-mass spectrometry (LC-MS) analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role associated with biofuel development. PMID:23176123

  10. Determination of the separate lipid and protein profile structures derived from the total membrane profile structure or isolated sarcoplasmic reticulum via x-ray and neutron diffraction

    Sarcoplasmic reticulum (SR) membranes were prepared to contain biosynthetically deuterated SR phospholipids utilizing specific and general phospholipid exchange proteins (PLEP). Functional measurements and freeze fracture on SR dispersions and x-ray diffraction of hydrated oriented membrane multilayers revealed that the exchanged SR membranes were very similar to unexchanged SR membranes. Low resolution (28-A) neutron diffraction studies utilizing SR membranes exchanged with either protonated or perdeuterated SR phospholipids allowed direct determination of the lipid profile within the isolated SR membrane at two different unit cell repeat distances. These lipid profile structures were found to be highly asymmetric regarding the conformation of the fatty acid chain extents and compositional distribution of phospholipid molecules in the inner vs. outer monolayer of the SR membrane bilayer. The relatively high resolution (11-A) electron-density profile from x-ray diffraction was decomposed by utilizing the asymmetry in the number of phospholipid molecules residing in the inner vs. outer monolayer of the SR lipid bilayer as obtained from the neutron diffraction study. To our knowledge, this represents the first direct determination of a lipid bilayer profile structure within an isolated membrane system

  11. SSpro/ACCpro 5: almost perfect prediction of protein secondary structure and relative solvent accessibility using profiles, machine learning and structural similarity

    Magnan, Christophe N.; Baldi, Pierre

    2014-01-01

    Motivation: Accurately predicting protein secondary structure and relative solvent accessibility is important for the study of protein evolution, structure and function and as a component of protein 3D structure prediction pipelines. Most predictors use a combination of machine learning and profiles, and thus must be retrained and assessed periodically as the number of available protein sequences and structures continues to grow.

  12. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

  13. Early embryonic gene expression profiling of zebrafish prion protein (Prp2 morphants.

    Rasoul Nourizadeh-Lillabadi

    Full Text Available BACKGROUND: The Prion protein (PRNP/Prp plays a crucial role in transmissible spongiform encephalopathies (TSEs like Creutzfeldt-Jakob disease (CJD, scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. METHODOLOGY/PRINCIPAL FINDINGS: The zebrafish (Danio rerio genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. CONCLUSIONS/SIGNIFICANCE: The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development.

  14. Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients.

    Liesbeth Bieghs

    Full Text Available Insulin-like growth factor (IGF signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM. In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6, leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17, monoclonal gammopathy of undetermined significance (MGUS (n = 37, and control individuals (n = 15, using ELISA (IGFs and 125I-IGF1 Western Ligand Blotting (IGFBPs. MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold and decrease in intact IGFBP-3 (0.6-0.5 fold in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.

  15. Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients.

    Bieghs, Liesbeth; Brohus, Malene; Kristensen, Ida B; Abildgaard, Niels; Bøgsted, Martin; Johnsen, Hans E; Conover, Cheryl A; De Bruyne, Elke; Vanderkerken, Karin; Overgaard, Michael T; Nyegaard, Mette

    2016-01-01

    Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration. PMID:27111220

  16. Comparison of protein expression profile changes in human fibroblasts induced by low doses of gamma rays and energetic protons

    Zhang, Ye; Clement, Jade; Gridley, Diala; Rohde, Larry; Wu, Honglu

    Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposure of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to more than 200 proteins (or modified proteins) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured till fully confluent and then exposed to 2 cGy of gamma rays at either low (1 cGy/hr) or high (0.2 Gy/min) dose-rate, or to 2 cGy of 150 MeV protons at high dose-rate. The proteins were isolated at 2 and 6 hours after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loaded onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. Comparison of the overall protein expression profiles in gamma-irradiated cells showed significantly higher inductions at the high dose-rate than at the low dose-rate. The protein profile in cells after the proton exposure showed a much earlier induction pattern in comparison to both the high and low dose-rate gamma exposures. The same expression patterns were also found in individual cell signaling cascades. At 6 hours post irradiation, high dose-rate gamma rays induced cellular protein level changes (ratio to control ˜2) mostly in apoptosis, cell cycle and cytoskeleton, while low dose-rate gamma rays induced similar changes with smaller fold-change values. In comparison, protons induced

  17. ''Activity based coasting'' in radiology

    Background: The introduction of diagnosis related groups for reimbursement of hospital services in Germany (g-drg) demands for a reconsideration of utilization of radiological products and costs related to them.Methods: Traditional cost accounting as approach to internal, department related budgets are compared with the accounting method of activity based costing (ABC). The steps, which are necessary to implement ABC in radiology are developed.Conclusions: The introduction of a process-oriented cost analysis is feasible for radiology departments. ABC plays a central role in the set-up of decentralized controlling functions within this institutions. The implementation seems to be a strategic challenge for department managers to get more appropriate data for adequate enterprise decisions. The necessary steps of process analysis can be used for other purposes (Certification, digital migration) as well. (orig.)

  18. 3PFDB - A database of Best Representative PSSM Profiles (BRPs of Protein Families generated using a novel data mining approach

    Shameer Khader

    2009-12-01

    Full Text Available Abstract Background Protein families could be related to each other at broad levels that group them as superfamilies. These relationships are harder to detect at the sequence level due to high evolutionary divergence. Sequence searches are strongly directed and influenced by the best representatives of families that are viewed as starting points. PSSMs are useful approximations and mathematical representations of protein alignments, with wide array of applications in bioinformatics approaches like remote homology detection, protein family analysis, detection of new members and evolutionary modelling. Computational intensive searches have been performed using the neural network based sensitive sequence search method called FASSM to identify the Best Representative PSSMs for families reported in Pfam database version 22. Results We designed a novel data mining approach for the assessment of individual sequences from a protein family to identify a single Best Representative PSSM profile (BRP per protein family. Using the approach, a database of protein family-specific best representative PSSM profiles called 3PFDB has been developed. PSSM profiles in 3PFDB are curated using performance of individual sequence as a reference in a rigorous scoring and coverage analysis approach using FASSM. We have assessed the suitability of 10, 85,588 sequences derived from seed or full alignments reported in Pfam database (Version 22. Coverage analysis using FASSM method is used as the filtering step to identify the best representative sequence, starting from full length or domain sequences to generate the final profile for a given family. 3PFDB is a collection of best representative PSSM profiles of 8,524 protein families from Pfam database. Conclusion Availability of an approach to identify BRPs and a curated database of best representative PSI-BLAST derived PSSMs for 91.4% of current Pfam family will be a useful resource for the community to perform detailed and

  19. Protein profiles in cortical and nuclear regions of aged human donor lenses: A confocal Raman microspectroscopic and imaging study.

    Vrensen, Gijs F J M; Otto, Cees; Lenferink, Aufried; Liszka, Barbara; Montenegro, Gustavo A; Barraquer, Rafael I; Michael, Ralph

    2016-04-01

    A combination of Raman spectroscopy, imaging, hierarchical cluster analysis (HCA) and peak ratio analysis was used to analyze protein profiles in the superficial cortex (SC), deep cortex (DC) and nucleus of old human lenses with cortical, nuclear and mixed cataracts. No consistent differences were observed in protein spectra and after cluster analysis between the three locations irrespective of the presence or absence of cortical opacities and/or coloration. A sharp increase (∼15%-∼33%) in protein content from SC to DC, normal for human lenses, was found in 7 lenses. In 4 lenses, characterized by the absence of cortical opacities, the SC has a protein content of ∼35%. A significant increase in the disulfide-to-protein ratio is found only in the SC of the 7 cortical cataracts. No changes were found in sulfhydryl-to-protein ratio. The relative contents of α-helices and β-sheets increase from SC to nucleus. β-Sheets are more common in the SC of lenses with cortical cataract. The absence of significant and consistent changes in protein profiles between nucleus and cortex even in cases of severe coloration is not favoring the prevailing concept that ubiquitous protein oxidation is a key factor for age related nuclear (ARN) cataracts. The observations favor the idea that multilamellar bodies or protein aggregates at very low volume densities are responsible for the rise in Mie light scatter as a main cause of ARN cataracts leaving the short-range-order of the fiber cytoplasm largely intact. The absence of significant changes in the protein spectra of the deep cortical opacities, milky white as a result of the presence of vesicle-like features, indicate they are packed with relatively undisturbed crystallins. PMID:26611157

  20. Molecular profiling of signalling proteins for effects induced by the anti-cancer compound GSAO with 400 antibodies

    GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide) is a hydrophilic derivative of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO). It inhibits angiogenesis and tumour growth in mouse models and may be evaluated in a phase I clinical trial in the near future. Initial experiments have implicated GSAO in perturbing mitochondrial function. Other molecular effects of GSAO in human cells, for example on the phosphorylation of proteins, are still largely unknown. Peripheral white blood cells (PWBC) from healthy volunteers were isolated and used to profile effects of GSAO vs. a control compound, GSCA. Changes in site-specific phosphorylations, other protein modifications and expression levels of many signalling proteins were analysed using more than 400 different antibodies in Western blots. PWBC were initially cultured in low serum conditions, with the aim to reduce basal protein phosphorylation and to increase detection sensitivity. Under these conditions pleiotropic intracellular signalling protein changes were induced by GSAO. Subsequently, PWBC were cultured in 100% donor serum to reflect more closely in vivo conditions. This eliminated detectable GSAO effects on most, but not all signalling proteins analysed. Activation of the MAP kinase Erk2 was still observed and the paxillin homologue Hic-5 still displayed a major shift in protein mobility upon GSAO-treatment. A GSAO induced change in Hic-5 mobility was also found in endothelial cells, which are thought to be the primary target of GSAO in vivo. Serum conditions greatly influence the molecular activity profile of GSAO in vitro. Low serum culture, which is typically used in experiments analysing protein phosphorylation, is not suitable to study GSAO activity in cells. The signalling proteins affected by GSAO under high serum conditions are candidate surrogate markers for GSAO bioactivity in vivo and can be analysed in future clinical trials. GSAO effects on Hic-5 in endothelial cells may

  1. Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1 in Maize

    Jin Yu

    2014-05-01

    Full Text Available Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L. and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1; which did not carry a gametophytic factor and W22 (Ga1, a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1 and W22 (Ga1. A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1 and W22 (Ga1 whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1 and W22 (Ga1. However, in pollen, 2 proteins were identified only in the W22 (ga1 and 12 proteins only in the W22 (Ga1 whereas 10 proteins were confirmed from the both of W22 (ga1 and W22 (Ga1. In contrary, 10 proteins were appeared only in the pistil of W22 (ga1 and 7 proteins from W22 (Ga1 while 3 proteins confirmed in the both of W22 (ga1 and W22 (Ga1. Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize.

  2. Identification and Expression Profile Analysis of Odorant Binding Proteins in the Oriental Fruit Fly Bactrocera dorsalis

    Hongyu Zhang

    2013-07-01

    Full Text Available Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10 are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies.

  3. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice

    Yi-Ming Chen

    2016-02-01

    Full Text Available Exercise and fitness training programs have attracted the public’s attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. Purpose: In this study, we designed a power exercise training (PET program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA-rich sake protein (SP to determine whether the supplement had a synergistic effect during PET and enhanced athletic performance and resistance to fatigue. Methods: Male ICR mice were divided into three groups (n = 8 per group for four-week treatment: sedentary controls with vehicle (SC, and PET and PET groups with SP supplementation (3.8 g/kg, PET + SP. Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time as well as changes in body composition and anti-fatigue activity levels of serum lactate, ammonia, glucose, and creatine kinase (CK after a 15-min swimming exercise. The biochemical parameters were measured at the end of the experiment. Results: four-week PET significantly increased grip strength and exhaustive swimming time and decreased epididymal fat pad (EFP weight and area. Levels of aspartate aminotransferase (AST, alanine aminotransferase (ALT, creatinine, and uric acid (UA were significantly increased. PET + SP supplementation significantly decreased serum lactate, ammonia and CK levels after the 15-min swimming exercise. The resting serum levels of AST, ALT, CREA and UA were all significantly decreased with PET + SP. Conclusion: The PET program could increase the exercise performance and modulate the body composition of mice. PET with SP conferred better anti-fatigue activity, improved biochemical profiles, and may be an effective ergogenic aid in strength training.

  4. Potential Biomarkers Found by Protein Profiling May Provide Insight for the Macrovascular Pathogenesis of Diabetes Mellitus

    William C. S. Cho

    2006-01-01

    Full Text Available Diabetes mellitus (DM is an alarming threat to health of mankind, yet its pathogenesis is unclear. The purpose of this study was to find potential biomarkers to serve as indicators for the pathogenesis of DM in a time course manner. Based on our previous findings that oxidative stress occurred at week 8, aorta lysate and sera of 102 streptozotocin (STZ-induced diabetic and 85 control male Sprague-Dawley rats were obtained at the 4th, 8th and 12th week after STZ injection. The protein profiles were studied employing surface-enhanced laser desorption/ionization time-of-flight mass spectrometry technology in attomole sensitivity range. In the aorta, a multiple biomarker panel was discovered at the 4th week. At the 8th week, 4 biomarkers were found, while at the 12th week, 3 biomarkers were identified. In the sera, a triplet of 3 peaks and 2 biomarkers were all discovered to have 100% classification accuracy rate to differentiate the DM and control groups at all time intervals. Besides, 2 biomarkers were also found to have high classification value at week 12. Comparing the aorta and sera from DM and non-DM rats, a bundle of potential biomarkers with significant changes in peak intensities and high classification values were found. Two of the serum biomarkers matched with islet amyloid polypeptide and resistin in the SWISS-PROT knowledgebase. Validation has been conducted using immunoassay kits. These potential biomarkers may provide valuable insight on the pathogenesis of DM and macrovascular complications.

  5. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

    Thiruvengadam Raguchander

    2009-12-01

    Full Text Available Abstract Background Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  6. Nutritional Composition and Protein Profile of Goat Yogurt PE with Double Culture between Streptococcus thermophilus and Lactobacilus species

    Ismi Kurnia Budiarti

    2013-08-01

    Full Text Available Aim: The aims of this study are to characterize the nutrient compositions and protein profiles of Etawah breed (PE goat yogurt fermented by double cultures . Material and Methods: To accomplish this, we used goat and bovine milk in five treatment groups: (1 fresh milk bovine, (2 goat,(3 milk fermented by L. acidophilus and S. thermophilus (LA + ST, (4 L. bulgaricus and S. thermophilus (LB+ ST,and (5 a comersial mixture. PE goat milk was fermented using 2.5% starting bacterial concentrations at 45oC with a pH ranging from 4.5 to 6.6. Nutrient compositions were measured by proximate analysis.SDS PAGE was conducted using 15% separating and 3% stacking gels. To measure the density of protein bands, we used QuantityOne software. Results: Our results indicated that LA+ST and LB+ST treatments had higher levels of lipids than the control treatment. Conversely, both strain combinations had lower levels of proteins than the control. Organoleptic testing suggests that many attributes (e.g., colour, taste, smell, texture and viscosity differ significantly from the control. Protein profiles revealed that while the LB + ST and commercial cultures contained proteins with a molecular weight of 36 kDa, the LA + ST cultures did not appear to possess this protein.Based on the molecular weight, we suggest that this protein is in the alpha casein group. Conclusion: The protein composition of fermented goat and bovine milk is similiar, but the absence of band with molecular weight 36 kDa from goat milk, LB+ST and mix comercial. [Cukurova Med J 2013; 38(4.000: 681-686

  7. Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

    Jafari, Mohieddin; Primo, Vincent; Smejkal, Gary B.; Moskovets, Eugene V.; Kuo, Winston P.; Ivanov, Alexander R.

    2012-01-01

    Fractionation of complex samples at the cellular, subcellular, protein or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e., 1-D SDS PAGE, preparative 1-D SDS PAGE, isoelectric focusing in immobilized pH gradients (IEF-IPG), and 2-D PAGE in their performance as fractionation approaches in nanoLC-ESI-MS/MS analysis of a mi...

  8. Expression profile and subcellular location of the plasmid-encoded virulence (Spv) proteins in wild-type Salmonella dublin.

    El-Gedaily, A; Paesold, G; Krause, M.

    1997-01-01

    The plasmid-encoded virulence genes (spvABCD) in nontyphoid Salmonella strains mediate lethal infections in a variety of animals. Previous studies have shown that these genes are transcriptionally regulated by stationary-phase growth. We studied the expression profile and the subcellular locations of the SpvABCD proteins in wild-type S. dublin by using polyclonal antibodies against SpvA, SpvB, SpvC, and SpvD. The cellular levels of the individual proteins were determined during growth by quan...

  9. Blood profiling of proteins and steroids during weight maintenance with manipulation of dietary protein level and glycaemic index

    Wang, Ping; Holst, Claus; Astrup, Arne;

    2012-01-01

    ), evenly selected from four dietary groups that varied in protein and GI levels. The blood concentrations of twenty-nine proteins and three steroid hormones were measured. The changes in analytes during weight maintenance largely correlated negatively with the changes during weight loss, with some...

  10. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling.

    Sasisekhar Bennuru

    Full Text Available Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf, L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm genome. Not surprisingly, the majority (160/274 of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase, MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

  11. Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

    Ozer, Abdullah; Tome, Jacob M.; Friedman, Robin C.; Gheba, Dan; Schroth, Gary P.; Lis, John T.

    2015-01-01

    Because RNA-protein interactions play a central role in a wide-array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the High Throughput Sequencing-RNA Affinity Profiling (HiTS-RAP) assay, which couples sequencing on an Illumina GAIIx with the quantitative assessment of one or several proteins’ interactions with millions of different RNAs in a single experiment. We have success...

  12. IMPACT OF CO2 ENHANCEMENT ON PHOTOSYNTHESIS AND PROTEIN PROFILE -RESPONSE STUDIES WITH A CO2 RESPONSIVE BLACK GRAM GENOTYPE

    P. Sathish

    2014-09-01

    Full Text Available Black gram (Vigna mungo (L. Hepper var. IC-282009 - a highly CO2 responsive genotype for biomass and seed yield was grown in Open top chambers (OTCs under three levels of CO2 i.e. ambient (390 ppm and two elevated levels 550ppm and 700ppm to assess photosynthetic acclimation to elevated CO2. Net photosynthetic rate (PN, change in leaf soluble protein profile and leaf carbohydrate constituents such as total soluble sugars, reducing sugars and starch content in leaves was quantified at all three CO2 concentrations. Photosynthetic rate was enhanced by 78% and 30% at flowering stage with 550ppm and 700ppm CO2 as compared with ambient control. It was also observed a higher accumulation of starch, total soluble sugars and reducing sugars in leaves at elevated CO2 levels. However, the leaf protein content recorded a decrease and altered the profile of ploy peptides with enhanced CO2 levels. At elevated CO2 concentrations significant differences were observed in ploy peptide profile at vegetative and flowering stages, the intensity of 260 kDa poly peptide increased at vegetative stage, whereas 72 kDa polypeptide increased at flowering stage, while 52 kDa poly peptide decreased at both stages. Enhanced CO2 concentrations improved the PN though certain polypeptides of leaf protein are down regulated and necessitate further experimentation to confirm their involvement in responsiveness of the selected black gram genotype

  13. Changes in the protein expression profiles of the Hepa-T1 cell line when exposed to Cu2+.

    Chen, Dong-Shi; Chan, King Ming

    2009-09-14

    Copper is an essential element in a variety of biological processes, but it can be toxic when present in excessive amounts. The central regulators of cellular copper metabolism include copper-binding proteins, copper transporters, metal membrane active transporters and copper-dependent enzymes. However, the way in which cupric ions (Cu(2+)) cause cellular changes in proteins and lead to toxic effects is less well-known. The aim of this study is to identify the proteins related to Cu(2+) toxicity or detoxification mechanisms in tilapia (Oreochromis niloticus) using a proteomic approach. A cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa-T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2DE). It was found that Cu(2+) (120 and 300 microM) caused the differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions. PMID:19616320

  14. A secretory system for bacterial production of high-profile protein targets

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin;

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To...... improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer...... membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly...

  15. Introgressive hybridization and evolution of a novel protein phenotype: glue protein profiles in the nasuta–albomicans complex of Drosophila

    S. Aruna; H. A. Ranganath

    2006-04-01

    Glue proteins are tissue-specific proteins synthesized by larval salivary gland cells of Drosophila. In Drosophila nasuta nasuta and D. n. albomicans of the nasuta subgroup, the genes that encode the major glue protein fractions are X-linked. In the present study, these X-linked markers have been employed to trace the pattern of introgression of D. n. nasuta and D. n. albomicans genomes with respect to the major glue protein fractions in their interracial hybrids, called cytoraces. These cytoraces have inherited the chromosomes of both parents and have been maintained in the laboratory for over 400–550 generations. The analysis has revealed that cytoraces with D. n. albomicans X chromosome show either D. n. nasuta pattern or a completely novel pattern of glue protein fractions. Further, quantitative analysis also shows lack of correlation between the chromosomal pattern of inheritance and overall quantity of the major glue protein fractions in the cytoraces. Thus, in cytoraces the parental chromosomes are not just differentially represented but there is evidence for introgression even at the gene level.

  16. Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family

    Vinogradov Serge

    2007-10-01

    Full Text Available Abstract Background The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. In silico analysis of the genome of C. elegans identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in C. elegans. Results All 33 C. elegans putative globin genes are transcribed. The translated sequences have the essential signatures of single domain bona fide globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of C. elegans. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in daf-2(e1370/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates hif-1 transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of C. elegans, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect

  17. Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging.

    Ramallo Guevara, Carina; Philipp, Oliver; Hamann, Andrea; Werner, Alexandra; Osiewacz, Heinz D; Rexroth, Sascha; Rögner, Matthias; Poetsch, Ansgar

    2016-05-01

    The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a

  18. Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

    Mosley R Lee

    2008-09-01

    Full Text Available Abstract Background The ProteomeLab™ PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS patients before and during immunization with glatiramer acetate (GA in a clinical trial. Results The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab™ IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion We offer some insight into the strengths and limitations of this emerging proteomic platform.

  19. Protein Profiling of Human Nonpigmented Ciliary Epithelium Cell Secretome: The Differentiation Factors Characterization for Retinal Ganglion Cell line

    Ming-Hui Yang

    2011-01-01

    Full Text Available The purpose of this paper was to characterize proteins secreted from the human nonpigmented ciliary epithelial (HNPE cells, which have differentiated a rat retinal ganglion cell line, RGC-5. Undifferentiated RGC-5 cells have been shown to express several marker proteins characteristic of retinal ganglion cells. However, RGC-5 cells do not respond to N-methyl-D aspartate (NMDA, or glutamate. HNPE cells have been shown to secrete numbers of neuropeptides or neuroproteins also found in the aqueous humor, many of which have the ability to influence the activity of neuronal cells. This paper details the profile of HNPE cell-secreted proteins by proteomic approaches. The experimental results revealed the identification of 132 unique proteins from the HNPE cell-conditioned SF-medium. The biological functions of a portion of these identified proteins are involved in cell differentiation. We hypothesized that a differentiation system of HNPE cell-conditioned SF-medium with RGC-5 cells can induce a differentiated phenotype in RGC-5 cells, with functional characteristics that more closely resemble primary cultures of rat retinal ganglion cells. These proteins may replace harsh chemicals, which are currently used to induce cell differentiation.

  20. Tumor suppressor gene NGX6 induces changes in protein expression profiles in colon cancer HT-29 cells

    Yu Li; Yuan Luo; Xiaoyan Wang; Shourong Shen; Haibo yu; Jing Yang; Zheng Su

    2012-01-01

    Nasopharyngeal carcinoma-associated gene 6 (NGX6;syn.transmembrane protein 8B,TMEM8B) is a recently identified tumor suppressor gene.The underlying mechanisms by which the gene inhibits tumor development are not completely known.To further understand the function of the gene's protein product NGX6,in the present study,we employed two-dimensional difference gel electrophoresis to analyze the protein expression profiles of colon cancer HT-29 cells stably transfected with the gene NGX6.The differentially expressed proteins were selected and identified by matrix-assisted laser desorption/ionization coupled with time-of-flight tandem mass spectrometry.The results showed that 12 proteins were down-regulated and 4 were up-regulated in NGX6-transfected HT-29 cells,compared with vector-transfected HT-29 cells.The MS results were verified by western blot.Bioinformatic analysis showed that these proteins are involved in cell proliferation,metastasis,apoptosis,cytoskeletal structure,metabolism,and signal transduction,suggesting that NGX6 may inhibit colon cancer through the regulation of these biological processes.

  1. Evidence of prognostic relevant expression profiles of heat-shock proteins and glucose-regulated proteins in oesophageal adenocarcinomas.

    Julia Slotta-Huspenina

    Full Text Available A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs and glucose-regulated proteins (GRPs are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p-HSP27((Ser15, p-HSP27((Ser78, p-HSP27((Ser82, HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA, immunohistochemistry (IHC and real-time quantitative RT-PCR (qPCR. Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27((Ser15, Ser78, Ser82 and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015 and multivariate analysis (p = 0.029. Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.

  2. Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

    Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

    2013-11-01

    Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. PMID:23769496

  3. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  4. Gene expression profiles of novel caprine placental prolactin-related proteins similar to bovine placental prolactin-related proteins

    Sato Eimei; Sasada Hiroshi; Abe Yasuyuki; Kizaki Keiichiro; Hosoe Misa; Takahashi Toru; Ushizawa Koichi; Hashizume Kazuyoshi

    2007-01-01

    Abstract Background This study reports the identification of a full-length cDNA sequence for two novel caprine prolactin-related proteins (cPRP1 and cPRP6), and their localization and quantitative expression in the placenta. Caprine PRPs are compared with known bovine PRPs. We examined their evolution and role in the ruminant placenta. Results Full-length cPRP1 and cPRP6 cDNA were cloned with a 717- and 720- nucleotide open-reading frame corresponding to proteins of 238 and 239 amino acids. T...

  5. Gene expression profiles of novel caprine placental prolactin-related proteins similar to bovine placental prolactin-related proteins

    Ushizawa, Koichi; Takahashi, Toru; Hosoe, Misa; Kizaki, Keiichiro; Abe, Yasuyuki; SASADA, Hiroshi; SATO, Eimei; Hashizume, Kazuyoshi

    2007-01-01

    Background This study reports the identification of a full-length cDNA sequence for two novel caprine prolactin-related proteins (cPRP1 and cPRP6), and their localization and quantitative expression in the placenta. Caprine PRPs are compared with known bovine PRPs. We examined their evolution and role in the ruminant placenta. Results Full-length cPRP1 and cPRP6 cDNA were cloned with a 717- and 720- nucleotide open-reading frame corresponding to proteins of 238 and 239 amino acids. The cPRP1 ...

  6. Myosin Binding Protein-C Slow: a multifaceted family of proteins with a complex expression profile in fast and slow twitch skeletal muscles

    Maegen A Ackermann

    2013-12-01

    Full Text Available Myosin Binding Protein-C slow (sMyBP-C comprises a complex family of proteins expressed in slow and fast type skeletal muscles. Similar to its fast and cardiac counterparts, sMyBP-C functions to modulate the formation of actomyosin cross-bridges, and to organize and stabilize sarcomeric A- and M-bands. The slow form of MyBP-C was originally classified as a single protein, however several variants encoded by the single MYBPC1 gene have been recently identified. Alternative splicing of the 5’ and 3’ ends of the MYBPC1 transcript has led to the differential expression of small unique segments interspersed between common domains. In addition, the NH2-terminus of sMyBP-C undergoes complex phosphorylation. Thus, alternative splicing and phosphorylation appear to regulate the functional activities of sMyBP-C. sMyBP-C proteins are not restricted to slow twitch muscles, but they are abundantly expressed in fast twitch muscles, too. Using bioinformatic tools, we herein perform a systematic comparison of the known human and mouse sMyBP-C variants. In addition, using single fiber westerns and antibodies to a common region of all known sMyBP-C variants, we present a detailed and comprehensive characterization of the expression profile of sMyBP-C proteins in the slow twitch soleus and the fast twitch flexor digitorum brevis (FDB mouse muscles. Our studies demonstrate for the first time that distinct sMyBP-C variants are co-expressed in the same fiber, and that their expression profile differs among fibers. Given the differential expression of sMyBP-C variants in single fibers, it becomes apparent that each variant or combination thereof may play unique roles in the regulation of actomyosin cross-bridges formation and the stabilization of thick filaments.

  7. Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse

    Yubo Ding; Yibo Wu; Rong Zeng; Kan Liao

    2012-01-01

    Lipid droplets in adipocytes serve as the principal longterm energy storage depot of animals.There is increasing recognition that lipid droplets are not merely a static neutral lipid storage site,but in fact dynamic and multifunctional organelles.Structurally,lipid droplet consists of a neutral lipid core surrounded by a phospholipid monolayer and proteins embedded in or bound to the phospholipid layer.Proteins on the surface of lipid droplets are crucial to droplet structure and dynamics.To understand the lipid droplet-associated proteome of primary adipocyte with a large central lipid droplet,lipid droplets of white adipose tissue from C57BL/6 mice were isolated.And the proteins were extracted and analyzed by liquid chromatography coupled with tandem mass spectrometry.A total of 193 proteins including 73 previously unreported proteins were identified.Furthermore,the isotope-coded affinity tags (ICAT) was used to compare the difference of lipid droplet-associated proteomes between the normal lean and the high-fat diet-induced obese C57BL/6 mice.Of 23proteins quantified by ICAT analysis,3 proteins were upregulated and 4 proteins were down-regulated in the lipid droplets of adipose tissue from the obese mice.Importantly,two structural proteins of lipid droplets,perilipin A and vimentin,were greatly reduced in the lipid droplets of the adipose tissue from the obese mice,implicating reduced protein machinery for lipid droplet stability.

  8. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  9. The Development of an Angiogenic Protein "Signature" in Ovarian Cancer Ascites as a Tool for Biologic and Prognostic Profiling.

    Trachana, Sofia-Paraskevi; Pilalis, Eleftherios; Gavalas, Nikos G; Tzannis, Kimon; Papadodima, Olga; Liontos, Michalis; Rodolakis, Alexandros; Vlachos, Georgios; Thomakos, Nikolaos; Haidopoulos, Dimitrios; Lykka, Maria; Koutsoukos, Konstantinos; Kostouros, Efthimios; Terpos, Evagelos; Chatziioannou, Aristotelis; Dimopoulos, Meletios-Athanasios; Bamias, Aristotelis

    2016-01-01

    Advanced ovarian cancer (AOC) is one of the leading lethal gynecological cancers in developed countries. Based on the important role of angiogenesis in ovarian cancer oncogenesis and expansion, we hypothesized that the development of an "angiogenic signature" might be helpful in prediction of prognosis and efficacy of anti-angiogenic therapies in this disease. Sixty-nine samples of ascitic fluid- 35 from platinum sensitive and 34 from platinum resistant patients managed with cytoreductive surgery and 1st-line carboplatin-based chemotherapy- were analyzed using the Proteome ProfilerTM Human Angiogenesis Array Kit, screening for the presence of 55 soluble angiogenesis-related factors. A protein profile based on the expression of a subset of 25 factors could accurately separate resistant from sensitive patients with a success rate of approximately 90%. The protein profile corresponding to the "sensitive" subset was associated with significantly longer PFS (8 [95% Confidence Interval {CI}: 8-9] vs. 20 months [95% CI: 15-28]; Hazard ratio {HR}: 8.3, pAOC, which can be used, after appropriate validation, as a prognostic marker and a tool for selection for anti-angiogenic therapies. PMID:27258020

  10. Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

    Nguyen, Hung T.; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois; Case, David A.

    2014-12-01

    A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb+ and Sr2+) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein-Zernike equations, with results from the Kovalenko-Hirata closure being closest to experiment for the cases studied here.

  11. Cellular fatty acid composition, protein profile and antimicrobial activity of Bacillus sp., isolated from fish gut

    Pushparaj Sujith; Baskaran Rohini; Singaram Jayalakshmi

    2014-01-01

    Objective: To purify and partially characterize the antimicrobial compounds from bacteriaBacillus sp., isolated from fish gut. Methods: Protein and fatty acids were isolated from the bacteria and checked for the presence of antibacterial activity. Protein has been purified to apparent homogeneity from the supernatants of culture by means of ammonium sulphate precipitation followed by dialysis. Fourier transform infrared spectroscopy analyses were performed for proteins to identify the functional groups.Results:sulfate polyacrylamide gel electrophoresis. Fatty acids were extracted and subjected to gas chromatographic analysis.Conclusions:Protein showed an apparent molecular mass 56, 47 and 39 kDa on sodium dodecyl acids and proteins which holds promise for the development of new drugs. The antimicrobial activity of the bacteria might be due to the presence of fatty acids and proteins which holds promise for the development of new drugs.

  12. A secretory system for bacterial production of high-profile protein targets

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin; Berthelsen, Jens; Weigelt, Johan; Gräslund, Susanne; Sundström, Michael

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A co...

  13. Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni

    Silveira, da, Fabio Land; Baumgärtner, M.; Rombouts, F. M.; Abee, T.

    2004-01-01

    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of O. oeni cells stressed with 12% ethanol ...

  14. Effect of Adaptation to Ethanol on Cytoplasmic and Membrane Protein Profiles of Oenococcus oeni

    2004-01-01

    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of O. oeni cells stressed with 12% ethanol ...

  15. EvoDesign: de novo protein design based on structural and evolutionary profiles

    Mitra, Pralay; Shultis, David; Zhang, Yang

    2013-01-01

    Protein design aims to identify new protein sequences of desirable structure and biological function. Most current de novo protein design methods rely on physics-based force fields to search for low free-energy states following Anfinsen’s thermodynamic hypothesis. A major obstacle of such approaches is the inaccuracy of the force field design, which cannot accurately describe the atomic interactions or distinguish correct folds. We developed a new web server, EvoDesign, to design optimal prot...

  16. Influence of various nitrogen applications on protein and amino acid profiles of amaranth and quinoa.

    Thanapornpoonpong, Sa-nguansak; Vearasilp, Suchada; Pawelzik, Elke; Gorinstein, Shela

    2008-12-10

    The effect of nitrogen application levels (0.16 and 0.24 g N kg(-1) soil) on seed proteins and their amino acid compositions of amaranth (Amaranthus spp.) and quinoa (Chenopodium quinoa Willd) was studied. Total proteins of amaranth and quinoa had high contents of lysine (6.3-8.2 g 100 g(-1) protein) but low contents of methionine (1.2-1.8 g 100 g(-1) protein). Seed proteins were fractionated on the basis of different solubility in water, saline, and buffer as albumin-1 (Albu-1), albumin-2 (Albu-2), globulin (Glob), and glutelin (Glu) and were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Albu-1 was high in lysine (5.4-8.6 g 100 g(-1) protein), while Albu-2, which is a part of storage proteins, had a high leucine content (7.2-8.9 g 100 g(-1) protein) as an effect of different nitrogen application levels. Glu fractions were well-balanced in their essential amino acids with the exception of methionine. In conclusion, nitrogen application can be used for the nutritional improvement in human diet by increasing and maintaining protein and essential amino acid contents. PMID:19006392

  17. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  18. Serum peptide/protein profiling by mass spectrometry provides diagnostic information independently of CA125 in women with an ovarian tumor

    Callesen, Anne; Madsen, Jonna S; Iachina, Maria; Vach, Werner; Kruse, Torben A; Jensen, Ole N; Mogensen, Ole

    2010-01-01

    investigated. Protein profiles of 113 serum samples from women with an ovarian tumor (54 malign and 59 benign) were generated using MALDI-TOF MS. A total of 98 peaks with a significant difference (p<0.01) in intensity between women with benign tumors/cysts and malignant ovarian tumors were identified. After...... protein profiling as a diagnostic tool in discrimination between benign ovarian tumors/cysts and malignant ovarian tumors. Additionally, the method provided diagnostic information independent of CA125.......In the present study, the use of a robust and sensitive mass spectrometry based protein profiling analysis was tested as diagnostic tools for women with an ovarian tumor. The potential additional diagnostic value of serum protein profiles independent of the information provided by CA125 were also...

  19. A study on the differential protein profiles in liver cells of heat stress rats with and without turpentine treatment

    Pen-huat Yap

    2009-01-01

    Full Text Available Abstract Background Heat stress (HS and related illnesses are a major concern in military, sports, and fire brigadiers. HS results in physiologic responses of increased temperature, heart rate and sweating. In heat stroke, inflammatory response plays an important role and it is evidenced that turpentine (T induced circulating inflammatory cytokines reduced survival rate and duration at 42°C. Here we report the alteration in the protein expression in liver cells upon HS with and without T treatment using two dimensional gel electrophoresis (2-DE, tryptic in-gel digestion and MALDI-TOF-MS/MS approaches Results The effects of HS and T treatments alone and a combined treatments (T+HS was performed in Wistar rat models. Proteomic analysis of liver in the HS and T+HS groups were analyzed compared to liver profiles of resting control and T treated groups. The study revealed a total of 25 and 29 differentially expressed proteins in the HS and T+HS groups respectively compared to resting control group. Fourteen proteins showed altered expression upon T treatment compared to resting control group. Proteins that are involved in metabolic and signal transduction pathways, defense, redox regulation, and cytoskeletal restructuring functions were identified. The altered expression of proteins reflected in 2D gels were corroborated by quantitative real time RT-PCR analysis of 8 protein coding genes representing metabolic and regulatory pathways for their expression and normalized with the house keeping gene β-actin Conclusion The present study has identified a number of differentially expressed proteins in the liver cells of rats subjected to T, HS and T+HS treatments. Most of these proteins are implicated in cell metabolism, as well as adaptive response to incurred oxidative stress and tissue damage due to T+HS and HS effects.

  20. C-reactive protein in patients with acute coronary syndrome: association with coronary markers, lipid profile and markers of coagulation

    To determine levels of C-reactive protein (CRP) and its association with coronary markers, lipid profile and markers of coagulation in patients of acute coronary syndrome (ACS). The study was conducted at Shifa college of Medicine and Shifa international hospital for a period of one year (November 2005-December 2006). Patients and Methods: Sixty nine age matched controls and 133 consecutive patients of ACS were included in the study. CRP were measured by immunoturbidometric method, MB fraction of creatine kinase (CK-MB) and Troponin-1 by micro-particle enzyme immunoassay, lipid levels by Colorimetric Enzymatic methods, platelets by celldyn and coagulation markers were measured by CA-50 Sysmax. At admission mean CRP levels, cardiac biomarkers, lipid profile and coagulation markers were significantly increased in patients of ACS versus controls. Within the patients of ACS the mean levels of CRP, CK-MB, Trop I, prothrombin time (PT) and activated partial thromboplastin time (Am) were significantly raised in patients with ST - elevation myocardial infarction (STEMI) and non STEMI (NSTEMI) versus patients of unstable angina (VA). Association between CRP levels and coronary markers, coagulation markers and lipid profile was found to be non significant. The CRP levels were increased in patients with ACS as compared to controls. The CRP levels were insignificantly correlated with coronary markers (CK-MB, Trop I), coagulation markers (platelet count, PT, Am), and lipid profile (cholesterol, triglyceride, HDL and LDL cholesterol) in patients with ACS. (author)

  1. Relations between the crude protein content and the amino acid profile of organically produced field beans (Vicia faba L.) and field peas (Pisum sativum L.)

    Witten, Stephanie; Aulrich, Karen

    2015-01-01

    In Organic Farming, grain legumes are important protein feedstuffs. There are hints indicating that the amino acid (AA) profile is affected by factors associated with varying crude protein (CP) contents of crops. The knowledge on this relationship between CP and AA profile needs to be extended to optimize feeding strategies for monogastrics as well as the selection of cultivars and varieties in fodder crop cultivation. Therefore, samples of 67 field beans (Vicia faba L.) and 86 field peas (Pi...

  2. Downregulation of the autophagy protein ATG-7 correlates with distinct sphingolipid profile in MCF-7 cells sensitized to photodamage

    Separovic, Duska; Kelekar, Ameeta; Tarca, Adi L.; Bielawski, Jacek; Kessel, David

    2009-06-01

    The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h) and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced, whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.

  3. Prioritizing orphan proteins for further study using phylogenomics and gene expression profiles in Streptomyces coelicolor

    Alam, Mohammad Tauqeer; Takano, Eriko; Breitling, Rainer

    2011-01-01

    Background: Streptomyces coelicolor, a model organism of antibiotic producing bacteria, has one of the largest genomes of the bacterial kingdom, including 7825 predicted protein coding genes. A large number of these genes, nearly 34%, are functionally orphan (hypothetical proteins with unknown funct

  4. Prioritizing orphan proteins for further study using phylogenomics and gene expression profiles in Streptomyces coelicolor.

    Alam, M.T.; Takano, E.; Breitling, R.

    2011-01-01

    ABSTRACT: BACKGROUND: Streptomyces coelicolor, a model organism of antibiotic producing bacteria, has one of the largest genomes of the bacterial kingdom, including 7825 predicted protein coding genes. A large number of these genes, nearly 34%, are functionally orphan (hypothetical proteins with unk

  5. FULL-LENGTH PEPTIDE ASSAY OF ANTIGENIC PROFILE OF ENVELOPE PROTEINS FROM SIBERIAN ISOLATES OF HEPATITIS C VIRUS

    A. A. Grazhdantseva

    2014-07-01

    Full Text Available Antigenic profiles of envelope glycoproteins of hepatitis C virus presented by three genotypes 1b, 2a/2c and 3a, which are most widespread in the territory of Russia and, in particular, in Novosibirsk, were studied using a panel of overlapping synthetic peptides. It was shown that highly immunogenic peptide epitopes of Е1 and Е2 proteins common for all HCV genotypes, are located in amino acid positions 250-260, 315-325 (Е1 protein, 390-400 (hypervariable region 1, 430-440, and 680-690 (Е2 protein. The greatest inter-genotypic differences were recorded in positions 280-290, 410-430 and 520-540. A novel antigenic determinant was detected in the region of aa 280-290 of the Е1 protein which was typical only for HCV 2a/2c genotype. A broad variation in the boundaries for the most epitopes suggests a high variability of the Е1 and Е2 viral proteins; however, a similar repertoire of antibodies induced by different HCV genotypes indicates to an opportunity of designing a new generation of cross-reactive HCV vaccines based on mapping of the E1 and E2 antigenic regions.

  6. Plasma proteome profiling of atherosclerotic disease manifestations reveals elevated levels of the cytoskeletal protein vinculin

    Kristensen, Lars P; Larsen, Martin Røssel; Mickley, Hans; Saaby, Lotte; Diederichsen, Axel Cosmus Pyndt; Lambrechtsen, Jess; Rasmussen, Lars M; Overgaard, Martin

    2014-01-01

    atherosclerotic diseases, and 4) individuals with an acute coronary syndrome. Immunoassays and SRM-MS were used for single patient verification of candidate proteins. Proteins involved in cardiovascular diseases i.e. serum amyloid protein A (SAA), C-reactive protein (CRP), and apolipoprotein(a) [apo(a)] displayed...... identify proteins with altered concentrations in plasma samples from four groups: 1) Individuals without cardiovascular symptoms and without the presence of coronary calcium, 2) individuals without cardiovascular symptoms, but with high amounts of coronary calcium, 3) individuals operated because of......Atherosclerosis is a chronic disease of the arterial wall that is recognized as the leading cause of mortality and morbidity worldwide. There is an eminent need for better biomarkers that can aid in patient care before the onset of the first cardiovascular event. We used quantitative proteomics to...

  7. Cellular fatty acid composition, protein profile and antimicrobial activity of Bacillus sp., isolated from fish gut

    Pushparaj Sujith

    2014-01-01

    Full Text Available Objective: To purify and partially characterize the antimicrobial compounds from bacteria Bacillus sp., isolated from fish gut. Methods: Protein and fatty acids were isolated from the bacteria and checked for the presence of antibacterial activity. Protein has been purified to apparent homogeneity from the supernatants of culture by means of ammonium sulphate precipitation followed by dialysis. Fourier transform infrared spectroscopy analyses were performed for proteins to identify the functional groups. Results: Protein showed an apparent molecular mass 56, 47 and 39 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fatty acids were extracted and subjected to gas chromatographic analysis. Conclusions: The antimicrobial activity of the bacteria might be due to the presence of fatty acids and proteins which holds promise for the development of new drugs.

  8. Nutritional Composition and Protein Profile of Goat Yogurt PE with Double Culture between Streptococcus thermophilus and Lactobacilus species

    Budiarti, Ismi Kurnia; Padaga, Masdiana C.; Fatchiyah, Fatchiyah

    2013-01-01

    Aim: The aims of this study are to characterize the nutrient compositions and protein profiles of Etawah breed (PE) goat yogurt fermented by double cultures . Material and Methods: To accomplish this, we used goat and bovine milk in five treatment groups: (1) fresh milk bovine, (2) goat,(3) milk fermented by L. acidophilus and S. thermophilus (LA + ST), (4) L. bulgaricus and S. thermophilus (LB+ ST),and (5) a comersial mixture. PE goat milk was fermented using 2.5% starting bacterial con...

  9. Nutritional Composition and Protein Profile of Goat Yogurt PE with Double Culture between Streptococcus thermophilus and Lactobacilus species

    Ismi Kurnia Budiarti; Padaga, Masdiana C.; Fatchiyah Fatchiyah

    2013-01-01

    Aim: The aims of this study are to characterize the nutrient compositions and protein profiles of Etawah breed (PE) goat yogurt fermented by double cultures . Material and Methods: To accomplish this, we used goat and bovine milk in five treatment groups: (1) fresh milk bovine, (2) goat,(3) milk fermented by L. acidophilus and S. thermophilus (LA + ST), (4) L. bulgaricus and S. thermophilus (LB+ ST),and (5) a comersial mixture. PE goat milk was fermented using 2.5% starting bacterial concentr...

  10. Profile and Functional Properties of Seed Proteins from Six Pea (Pisum sativum Genotypes

    Nikola Ristic

    2010-12-01

    Full Text Available Extractability, extractable protein compositions, technological-functional properties of pea (Pisum sativum proteins from six genotypes grown in Serbia were investigated. Also, the relationship between these characteristics was presented. Investigated genotypes showed significant differences in storage protein content, composition and extractability. The ratio of vicilin:legumin concentrations, as well as the ratio of vicilin + convicilin: Legumin concentrations were positively correlated with extractability. Our data suggest that the higher level of vicilin and/or a lower level of legumin have a positive influence on protein extractability. The emulsion activity index (EAI was strongly and positively correlated with the solubility, while no significant correlation was found between emulsion stability (ESI and solubility, nor between foaming properties and solubility. No association was evident between ESI and EAI. A moderate positive correlation between emulsion stability and foam capacity was observed. Proteins from the investigated genotypes expressed significantly different emulsifying properties and foam capacity at different pH values, whereas low foam stability was detected. It appears that genotype has considerable influence on content, composition and technological-functional properties of pea bean proteins. This fact can be very useful for food scientists in efforts to improve the quality of peas and pea protein products.

  11. Protein expression profiling during chick retinal maturation: a proteomics-based approach

    Stitt Alan W

    2008-12-01

    Full Text Available Abstract Background The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E development commencing at day 12 through 13, 17, 19 and post-hatch (P 1 and 33 days. Results 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF, PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line, nucleophosmin 1 (NPM1, dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP, beta-synuclein and enhancer of rudimentary homologue. Conclusion This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

  12. Proteomic Profiling of Differentially Expressed Proteins from Bax inhibitor-1 Knockout and Wild Type Mice

    Li, Bo; John C Reed; Kim, Hyung-Ryong; Chae, Han-Jung

    2012-01-01

    Bax inhibitor-1 (BI-1) is an anti-apoptotic protein located in the endoplasmic reticulum (ER). The role of BI-1 has been studied in different physiopathological models including ischemia, diabetes, liver regeneration and cancer. However, fundamental knowledge about the effects of BI-1 deletion on the proteome is lacking. To further explore this protein, we compared the levels of different proteins in bi-1−/− and bi-1+/+ mouse tissues by two-dimensional electrophoresis (2-DE) and mass spectrom...

  13. SDS-Page Seed Storage Protein Profiles in Chili Peppers (Capsicum L.)

    Owk ANIEL KUMAR; Sape SUBBA TATA

    2010-01-01

    Seed protein banding patterns (SDS-PAGE) were studied from eighteen genotypes of chili pepper (Capsicum L). A total of 21 protein polypeptide bands with molecular weight ranging from 18.6 to 72.0 kD were recorded. Among the genotypes �CA18�, �CA21� and �CA27� represented maximum number of protein bands (12). Band no. (11) and (5,12) are exclusive to C. annuum L. and C. frutescens L. genotypes respectively. Average percent similarity was highest (100%) between �CA2� and �CA8� genotypes and the...

  14. Effects of Regular Recreational Exercise Training on Serum ANGPTL3-Like Protein and Lipid Profile in Young Healthy Adults

    Smol Ewelina

    2015-12-01

    Full Text Available Evidence of the role of ANGPTL3, a liver-secreted glycoprotein, in serum lipid turnover, led us to hypothesize that this protein may be involved in modification of the lipid profile induced by exercise-training. Given the lack of data regarding this issue, the main goal of the present study was to investigate the effects of regular participation in a recreational physical activity program on serum ANGPTL3 and selected lipid profile measures in young, apparently healthy female and male adults. We compared serum ANGPTL3, lipid profile measures, common lipid ratios, the Atherogenic Index of Plasma (AIP and glucose in fasting blood samples derived from 22 active physical education students including active females (AF, N=6 and males (AM, N=16 with samples from 28 relatively sedentary agematched peers, including female (SF, N=9 and male (SM, N=19 individuals not involved in any regular physical conditioning program. Despite high inter-individual variability of serum ANGPTL3, there was a general tendency toward higher serum ANGPTL3 and HDL-C in women compared to men, but without significant differences related to their physical activity status. Based on both routine lipid profile measures and lipid ratios, all participants had normal lipid profiles, normal glycemia, as well as favorable anthropometric indices not suggesting increased cardiometabolic risk. However, lower levels of the TG/HDL-C ratio and AIP in physically active compared to relatively sedentary participants, reflecting the predominance of large, buoyant LDL particles, strongly support the view of beneficial healthpromoting effects of regular participation in recreational sport activities.

  15. Identification of a putative protein profile associating with tamoxifen therapy resistance in breast cancer

    Umar, Arzu; Kang, Hyuk; Timmermans, A. M.; Look, Maxime P.; Meijer-van Gelder, M. E.; den Bakker, Michael A.; Jaitly, Navdeep; Martens, John W.; Luider, Theo M.; Foekens, John A.; Pasa-Tolic, Ljiljana

    2009-06-01

    Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.

  16. Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  17. Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models

    Tumor and stromal cells secrete a variety of proteins acting as extracellular signals and creating a supportive microenvironment for tumor development, angiogenesis, and metastasis. We used the Luminex immunoassay platform (including MILLIPLEX® MAP cytokine/chemokine, bone metabolism, adipocyte, M...

  18. Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

    Ngara, Rudo; Ndimba, Roya; Borch-Jensen, Jonas;

    2012-01-01

    sorghum variety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with...... MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p...

  19. Changes in male rat urinary protein profile during puberty: a pilot study

    Vettorazzi, A; R. Wait; Nagy, J.; Monreal, JI; Mantle, P

    2013-01-01

    BACKGROUND: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication betw...

  20. Prediction of heme binding residues from protein sequences with integrative sequence profiles

    2012-01-01

    Background The heme-protein interactions are essential for various biological processes such as electron transfer, catalysis, signal transduction and the control of gene expression. The knowledge of heme binding residues can provide crucial clues to understand these activities and aid in functional annotation, however, insufficient work has been done on the research of heme binding residues from protein sequence information. Methods We propose a sequence-based approach for accurate prediction...

  1. Proteomic analysis and protein carbonylation profile in trained and untrained rat muscle

    F.Magherini; P.M. Abruzzo; Puglia, M.; Bini, L.; T. Gamberi; Esposito, F; A. Veicsteinas; Marini, M.; Fiorillo, C; Gulisano, M; Modesti, A

    2012-01-01

    Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in tra...

  2. Ribosome Profiling Provides Evidence that Large Noncoding RNAs Do Not Encode Proteins

    Guttman, Mitchell; Russell, Pamela; Ingolia, Nicholas T.; Weissman, Jonathan S.; Lander, Eric S.; Lander, Eric S.

    2013-01-01

    Large noncoding RNAs are emerging as an important component in cellular regulation. Considerable evidence indicates that these transcripts act directly as functional RNAs rather than through an encoded protein product. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. Here, we show that classical noncoding RNAs and 5′ UTRs show the same ribosome occupancy a...

  3. Gene expression profiles and phosphorylation patterns of AMP-activated protein kinase subunits in various mesenchymal cell types

    Wang Yugang; Fan Qiming; Ma Rui; Lin Wentao; Tang Tingting

    2014-01-01

    Background Recent studies on bone have shown an endocrine role of the skeleton,which could be impaired in various human diseases,including osteoporosis,obesity,and diabetes-associated bone diseases.As a sensor and regulator of energy metabolism,AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism.The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.Methods Reverse transcription-polymerase chain reaction (PCR) for relative quantification,real-time PCR for absolute quantification,and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types,including primary human mesenchymal stem cells (hMSCs) and hFOB,Saos-2,C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells.Results AMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types.AMPKY1 mRNA was abundantly expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 but not detected in human-derived cell types.AMPKY2 mRNA was mildly expressed in all cell types.AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells.AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2,in which AMPKβ2 protein overwhelmed AMPKβ1 expression.AMPKy1 and AMPKY2 proteins were expressed in C3H/10T1/2,MC3T3-E1,3T3-L1,and C2C12 cells and only AMPKY2 protein was expressed in hMSCs,hFOB and Saos2 cells.AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.Conclusion The combination of AMPK α,β,and Y subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.

  4. Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don Endl

    Chen Yu-Ting

    2010-12-01

    Full Text Available Abstract Background Restoration of rooting competence is important for rejuvenation in Sequoia sempervirens (D. Don Endl and is achieved by repeatedly grafting Sequoia shoots after 16 and 30 years of cultivation in vitro. Results Mass spectrometry-based proteomic analysis revealed three proteins that differentially accumulated in different rejuvenation stages, including oxygen-evolving enhancer protein 2 (OEE2, glycine-rich RNA-binding protein (RNP, and a thaumatin-like protein. OEE2 was found to be phosphorylated and a phosphopeptide (YEDNFDGNSNVSVMVpTPpTDK was identified. Specifically, the protein levels of OEE2 increased as a result of grafting and displayed a higher abundance in plants during the juvenile and rejuvenated stages. Additionally, SsOEE2 displayed the highest expression levels in Sequoia shoots during the juvenile stage and less expression during the adult stage. The expression levels also steadily increased during grafting. Conclusion Our results indicate a positive correlation between the gene and protein expression patterns of SsOEE2 and the rejuvenation process, suggesting that this gene is involved in the rejuvenation of Sequoia sempervirens.

  5. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. PMID:27145233

  6. Protein Profiling of Gonads of Males and Sex Reversed Males in Nemacheilus angorae

    Maryam Cheraghzadeh

    2013-01-01

    Full Text Available In the present study a proteomics approach has been taken to analyze differential protein expression between mature male and sex reversed male of Nemacheilus angorae In regard to the fruitful studies of sex reversal in mammalian species and the fact that some major sex determination molecules are conserved among vertebrates, Nemacheilus angorae (Angorae loach seems to be a good model system in studying molecules involved in sex differentiation.N. angorae is a teleports fish exhibiting a spontaneous sex reversal (male to female pattern. The gonads of adult individuals were dissected and used for histological investigation and protein analysis. Proteins were next analyzed using two-dimensional gel electrophoresis and the distinguished spots have been compared in two experimental samples. Among them, 23 differentially expressed proteins spots were identified by MALDI-TOF/TOF analysis. Two spots in sex reversed testis with high score showed significant similarity to Vasa (assembling of the pole plasm and the pronuclear region of the oocyte and Proline 4-hydroxylase proteins. Vasa are involved in germ cell development both in invertebrates and vertebrates. This data could be considered as starting base for subsequent studies to identify proteins involved in sex reversal and differentiation at different stages of gonadal maturation in fish.

  7. Proteomic Profiling Reveals Upregulated Protein Expression of Hsp70 in Keloids

    Ju Hee Lee

    2013-01-01

    Full Text Available Background. The biochemical characteristics of keloid-derived fibroblasts differ from those of adjacent normal fibroblasts, and these differences are thought to be the cause of abnormal fibrosis. Therefore, we investigated the characteristic proteins that are differentially expressed in keloid-derived fibroblasts using proteomics tools. Objective. We attempted to investigate the novel proteins that play important roles in the pathophysiology of keloids. Methods. Proteomics analysis was performed to identify differentially expressed proteins in keloid-derived fibroblasts. Keloid-derived fibroblasts and adjacent normal fibroblasts were analyzed with 2-DAGE. We validated these proteins with immunoblot analysis, real-time RT-PCR, and immunohistochemistry. Results. Sixteen differentially expressed protein spots were identified in keloid-derived fibroblasts. Among them, heat shock protein 70 (Hsp70 was specifically upregulated in keloid-derived fibroblasts. Also, immunohistochemistry and western blot analysis revealed increased Hsp70, TGF-β, and PCNA expressions in keloids compared to normal tissue. Conclusion. Hsp70 is overexpressed in keloid fibroblasts and tissue. The overexpression of Hsp70 may be involved in the pathogenesis of keloids, and the inhibition of Hsp70 could be a new therapeutic tool for the treatment of keloids.

  8. Protein Profile of Human Lung Squamous Carcinoma Cell Line NCI-H226

    HAO ZHANG; NA LI; YUE CHEN; LING-YUN HUANG; YI-CHING WANG; GANG FANG; DA-CHENG HE; XUE-YUAN XIAO

    2007-01-01

    Objective To construct a database of human lung squamous carcinoma cell line NCI-H226 and to facilitate discovery of novel subtypes markers of lung cancer. Method Proteomic technique was used to analyze human lung squamous carcinoma cell line NCI-H226. The proteins of the NCI-H226 cells were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Results The results showed that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among three 2-D gels was 1.95±0.53 mm in the isoelectric focusing direction,and 1.73±0.45 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. One hundred and twenty-seven proteins, including enzymes, signal transduction proteins, structure proteins, transport proteins, etc. were characterized, of which, 29 identified proteins in NCI-H226 cells were reported for the first time to be involved in lung cancer carcinogenesis.Conclusion The information obtained from this study could provide some valuable clues for further study on the carcinogenetic mechanism of different types of lung cancer, and may help us to discover some potential subtype-specific biomarkers of lung cancer.

  9. Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets

    Stevenson, L.S.; McCullough, K.; Vincent, I.; Gilpin, D.F.; Summerfield, A.; Nielsen, Jens; McNeilly, F.; Adair, B.M.; Allan, G.M.

    2006-01-01

    The purpose of this study was to determine serum profiles of cytokines at a protein level and C-reactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a...

  10. Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

    Marius Schild

    2016-01-01

    Full Text Available Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n=19 and untrained (SED, n=17 individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

  11. The study of lipid profile, LP (a and electrolytes with oxidative stress, total protein and albumin in nephrotic syndrome

    Jyoti Dwivedi

    2014-02-01

    Full Text Available Nephrotic syndrome is characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia associated with peripheral edema. Recent observation revealed that serum albumin plays an important role in the host defense mechanism as it is one of the important antioxidants. Oxidative damage by free radicals has been implicated in kidney injury, especially in nephrotic syndrome (NS. Therefore, this study was carried out to investigate oxidant and lipoprotein (a status with protein and electrolytes in nephrotic syndrome patients. The blood samples were analyzed for quantitation of malondialdehyde as index of lipid peroxide, total antioxidant capacity, lipid profile, lipoprotein (a, electrolytes, total protein and albumin. Significantly increased levels of serum lipid peroxide, lipoprotein (a LDL, VLDL, Tcholand decreased levels of serum total antioxidant capacity and total protein and albumin were noticed in the patients with nephrotic syndrome as compared to control subjects. Electrolytes are variable Na was increased and potassium was decreased. However, significant positive correlation in lipid peroxide with lipoprotein (a,and total protein and albumin with total antioxidant capacity were observed. [Int J Res Med Sci 2014; 2(1.000: 62-66

  12. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    Karen Elizabeth Kempsell

    2015-08-01

    Full Text Available A commercial Bacillus anthracis (Anthrax whole genome protein microarray has been used to identify immunogenic Anthrax proteins using sera from groups of donors with (a confirmed B. anthracis naturally acquired cutaneous infection, (b confirmed B. anthracis intravenous drug use-acquired infection (c occupational exposure in a wool-sorters factory (d humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups.Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However a number of other chromosomally-located and plasmid encoded open reading frames were also recognised by infected or exposed groups in comparison to controls. Some of these antigens e.g. BA4182 are not recognised by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo and are not currently found in the UK licensed Anthrax Vaccine (AVP. These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis ‘infectome’. These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesised, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  13. Genome-Wide Profiling of RNA-Protein Interactions Using CLIP-Seq.

    Stork, Cheryl; Zheng, Sika

    2016-01-01

    UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein-RNA interactions in tissues and cells. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein-epitope tag is then used to immunoprecipitate the protein-RNA complexes. After stringent washing and gel separation the RBP-RNA complex is excised. The RBP is protease digested to allow purification of the bound RNA. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. Here, we describe the detailed procedure of iCLIP. PMID:26965263

  14. The levels of nitrite and nitrate, proline and protein profiles in tomato plants infected with pseudomonas syringae

    In this study, the contents of nitrite-nitrate and free L-proline, and pathogenesis-related (PR) proteins in tomato plants following inoculation with Pseudomonas syringae pv. tomato strain were examined. The results of the nitrite and nitrate indicated that there was a reduction in the levels of nitrate in the infected tomato plants through 1-8 study days, compared with the healthy plants. On the other hands, when the nitrite amounts increased in the first and second days, the nitrite concentrations reduced in infected plants at subsequent time periods, compared with uninfected plants. The accumulation of free proline increased in the infected plants, according to control plants. The whole-cell protein profiles displayed that the levels of the protein bands of molecular masses 204.6 kDa and 69.9 kDa significantly increased in infected and uninfected plants during 2-10 study days. In additionally, in the quantities of the protein bands of molecular weights 90.3 and 79.4 kDa were observed an increase in the infected and healthy plants after the fourth day. However, the protein band of molecular weight 54.3 kDa was visible only in uninfected plants for the fourth and eighth days. Finally, the study suggest that there were the sophisticate relationships among the proline accumulation, the conversion of nitrate to nitrite and the induction of PR protein genes in the regulation of defense mechanisms toward microbial invaders. Our results also indicated that the increases in nitrite and proline contents might be useful indicator for the response toward pathogen attacks. (author)

  15. Effects of the dietary protein level on the microbial composition and metabolomic profile in the hindgut of the pig.

    Zhou, Liping; Fang, Lingdong; Sun, Yue; Su, Yong; Zhu, Weiyun

    2016-04-01

    The aim of this study was to investigate the effects of a long-term low protein diet on the microbial composition and metabolomic profile in the hindgut of the pig. Thirty-six Duroc × Landrace × Large White growing barrows (70 days of age, 23.57 ± 1.77 kg) were randomly allocated to normal protein diet (NP) and low protein diet (LP) groups using a randomized block design. At the age of 170 days, the digesta in the hindguts of the pigs were collected for microbial and metabolomic analysis. The results showed that there were no significant differences in the average daily gain, average daily feed intake, or feed:gain ratio between the NP and LP groups. The concentrations of isobutyrate, isovalerate, and branched-chain fatty acids (BCFAs)/short-chain fatty acids (SCFAs) in the cecum decreased with the reduction of dietary protein. Pyrosequencing of the V1-V3 region of the 16S rRNA genes showed that LP diet significantly decreased the relative abundance of Lactobacillus in the cecum, and Streptococcus in the colon; however, the relative abundance of Prevotella and Coprococcus in the LP group was significantly higher than in the NP group in the cecum, and Sarcina, Peptostreptococcaceae incertae sedis, Mogibacterium, Subdoligranulum, and Coprococcus was higher in the colon. The gas chromatography-mass spectrometry (GC-MS) analysis showed that the dietary protein level mainly affected phenylalanine metabolism; glycine, serine, and threonine metabolism; the citrate cycle; pyruvate metabolism; and the alanine, aspartate, and glutamate metabolism. Moreover, the correlation analysis of the combined datasets revealed some potential relationships between the colonic metabolites and certain microbial species. These results suggest that a low protein diet may modulate the microbial composition and metabolites of the hindgut, without affecting the growth performance of pigs; however, potential roles of this modulation to the health of pigs remains unknown. PMID:26723572

  16. Comparisons of protein profiles of beech bark disease resistant and susceptible American beech (Fagus grandifolia

    Mason Mary E

    2013-01-01

    Full Text Available Abstract Background Beech bark disease is an insect-fungus complex that damages and often kills American beech trees and has major ecological and economic impacts on forests of the northeastern United States and southeastern Canadian forests. The disease begins when exotic beech scale insects feed on the bark of trees, and is followed by infection of damaged bark tissues by one of the Neonectria species of fungi. Proteomic analysis was conducted of beech bark proteins from diseased trees and healthy trees in areas heavily infested with beech bark disease. All of the diseased trees had signs of Neonectria infection such as cankers or fruiting bodies. In previous tests reported elsewhere, all of the diseased trees were demonstrated to be susceptible to the scale insect and all of the healthy trees were demonstrated to be resistant to the scale insect. Sixteen trees were sampled from eight geographically isolated stands, the sample consisting of 10 healthy (scale-resistant and 6 diseased/infested (scale-susceptible trees. Results Proteins were extracted from each tree and analysed in triplicate by isoelectric focusing followed by denaturing gel electrophoresis. Gels were stained and protein spots identified and intensity quantified, then a statistical model was fit to identify significant differences between trees. A subset of BBD differential proteins were analysed by mass spectrometry and matched to known protein sequences for identification. Identified proteins had homology to stress, insect, and pathogen related proteins in other plant systems. Protein spots significantly different in diseased and healthy trees having no stand or disease-by-stand interaction effects were identified. Conclusions Further study of these proteins should help to understand processes critical to resistance to beech bark disease and to develop biomarkers for use in tree breeding programs and for the selection of resistant trees prior to or in early stages of BBD

  17. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    Adriana FIORINI

    2016-01-01

    Full Text Available Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC and sub-minimal inhibitory concentration (sub-MIC of the butanolic extract (BUTE of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1, amino acid metabolism (ILV5, PDC11 and protein synthesis (ASC1 pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides, it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

  18. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  19. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    FIORINI, Adriana; ROSADO, Fabio Rogério; BETTEGA, Eliane Martins da Silva; MELO, Kátia Cristina Sibin; KUKOLJ, Caroline; BONFIM-MENDONÇA, Patrícia de Souza; SHINOBU-MESQUITA, Cristiane Suemi; GHIRALDI, Luciana Dias; CAMPANERUT, Paula Aline Zanetti; CAPOCI, Isis Regina Grenier; GODOY, Janine Silva Ribeiro; FERREIRA, Izabel Cristina Piloto; SVIDZINSKI, Terezinha Inez Estivalet

    2016-01-01

    Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds. PMID:27074319

  20. Identification and expression profiling of odorant binding proteins and chemosensory proteins between two wingless morphs and a winged morph of the cotton aphid Aphis gossypii glover.

    Shao-Hua Gu

    Full Text Available Insects interact with their environment and respond to the changes in host plant conditions using semiochemicals. Such ecological interactions are facilitated by the olfactory sensilla and the use of olfactory recognition proteins. The cotton aphid Aphis gossypii can change its phenotype in response to ecological conditions. They reproduce mainly as wingless asexual morphs but develop wings to find mates or new plant hosts under the influence of environmental factors such as temperature, plant nutrition and population density. Two groups of small soluble proteins, odorant binding proteins (OBPs and chemosensory proteins (CSPs are believed to be involved in the initial biochemical recognition steps in semiochemical perception. However, the exact molecular roles that these proteins play in insect olfaction remain to be discovered. In this study, we compared the transcriptomes of three asexual developmental stages (wingless spring and summer morphs and winged adults and characterised 9 OBP and 9 CSP genes. The gene structure analysis showed that the number and length of introns in these genes are much higher and this appears to be unique feature of aphid OBP and CSP genes in general. Another unique feature in aphids is a higher abundance of CSP transcripts than OBP transcripts, suggesting an important role of CSPs in aphid physiology and ecology. We showed that some of the transcripts are overexpressed in the antennae in comparison to the bodies and highly expressed in the winged aphids compared to wingless morphs, suggesting a role in host location. We examined the differential expression of these olfactory genes in ten aphid species and compared the expression profile with the RNA-seq analyses of 25 pea aphid transcriptome libraries hosted on AphidBase.

  1. Exploring the Plant–Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains

    Sultan, Abida; Andersen, Birgit; Svensson, Birte;

    2016-01-01

    Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant......-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation and...... included xylanases. The surface-associated proteomes showed elevated xylanolytic activity and contained several xylanases. Integration of proteomics with enzyme assays is a powerful tool for analysis and characterization of the interaction between microbial consortia and plants in their natural environment....

  2. Phytochemical-mediated Protein Expression Profiling and the Potential Applications in Therapeutic Drug Target Identifications.

    Wong, Fai-Chu; Tan, Siok-Thing; Chai, Tsun-Thai

    2016-07-29

    Many phytochemicals derived from edible medicinal plants have been investigated intensively for their various bioactivities. However, the detailed mechanism and their corresponding molecular targets frequently remain elusive. In this review, we present a summary of the research works done on phytochemical-mediated molecular targets, identified via proteomic approach. Concurrently, we also highlighted some pharmaceutical drugs which could be traced back to their origins in phytochemicals. For ease of presentation, these identified protein targets were categorized into two important healthcare-related fields, namely anti-bacterial and anti-cancer research. Through this review, we hope to highlight the usefulness of comparative proteomic as a powerful tool in phytochemical-mediated protein target identifications. Likewise, we wish to inspire further investigations on some of these protein targets identified over the last few years. With contributions from all researchers, the accumulative efforts could eventually lead to the discovery of some target-specific, low-toxicity therapeutic agents. PMID:26193174

  3. Prioritizing orphan proteins for further study using phylogenomics and gene expression profiles in Streptomyces coelicolor

    Takano Eriko

    2011-09-01

    Full Text Available Abstract Background Streptomyces coelicolor, a model organism of antibiotic producing bacteria, has one of the largest genomes of the bacterial kingdom, including 7825 predicted protein coding genes. A large number of these genes, nearly 34%, are functionally orphan (hypothetical proteins with unknown function. However, in gene expression time course data, many of these functionally orphan genes show interesting expression patterns. Results In this paper, we analyzed all functionally orphan genes of Streptomyces coelicolor and identified a list of "high priority" orphans by combining gene expression analysis and additional phylogenetic information (i.e. the level of evolutionary conservation of each protein. Conclusions The prioritized orphan genes are promising candidates to be examined experimentally in the lab for further characterization of their function.

  4. Chemically modified carbon nanotubes as material enhanced laser desorption ionisation (MELDI) material in protein profiling

    Biomarkers play a potential role in the early detection and diagnosis of a disease. Our aim is to derivatize carbon nanotubes for exploration of the differences in human body fluids e.g. serum, through matrix assisted laser desorption ionisation/time of flight mass spectrometry (MALDI/TOF-MS) that can be related to disease and subsequently to be employed in the biomarker discovery process. This application we termed as the material enhanced laser desorption ionisation (MELDI). The versatility of this technology is meant to increase the amount of information from biological samples on the protein level, which will have a major impact to serve the cause of diagnostic markers. Serum peptides and proteins are immobilized on derivatized carbon nanotubes, which function as binding material. Protein-loaded suspension is placed on a stainless steel target or buckypaper on aluminum target for direct analysis with MALDI-MS. The elution method to wash the bound proteins from carbon nanotubes was employed to compare with the direct analysis procedure. Elution is carried out by MALDI matrix solution to get them out of the entangled nanotubes, which are difficult to desorb by laser due to the complex nanotube structures. The advantage of these optimized methods compared to the conventional screening methods is the improved sensitivity, selectivity and the short analysis time without prior albumin and immunoglobulin depletion. The comparison of similarly modified diamond and carbon nanotubes exhibit differences in their nature to bind the proteins out of serum due to the differences in their physical characteristics. Infrared (IR) spectroscopy provided hint for the presence of tertiary amine peak at the crucial chemical step of iminodiacetic acid addition to acid chloride functionality on carbon nanotubes. Atomic absorption spectroscopy (AAS) was utilized to quantitatively measure the copper capacity of these derivatized carbon nanotubes which is a direct measure of capacity of

  5. Chemically modified carbon nanotubes as material enhanced laser desorption ionisation (MELDI) material in protein profiling

    Najam-ul-Haq, M. [Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck (Austria); Rainer, M. [Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck (Austria); Schwarzenauer, T. [Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck (Austria); Huck, C.W. [Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck (Austria)]. E-mail: christian.w.huck@uibk.ac.at; Bonn, G.K. [Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck (Austria)

    2006-03-02

    Biomarkers play a potential role in the early detection and diagnosis of a disease. Our aim is to derivatize carbon nanotubes for exploration of the differences in human body fluids e.g. serum, through matrix assisted laser desorption ionisation/time of flight mass spectrometry (MALDI/TOF-MS) that can be related to disease and subsequently to be employed in the biomarker discovery process. This application we termed as the material enhanced laser desorption ionisation (MELDI). The versatility of this technology is meant to increase the amount of information from biological samples on the protein level, which will have a major impact to serve the cause of diagnostic markers. Serum peptides and proteins are immobilized on derivatized carbon nanotubes, which function as binding material. Protein-loaded suspension is placed on a stainless steel target or buckypaper on aluminum target for direct analysis with MALDI-MS. The elution method to wash the bound proteins from carbon nanotubes was employed to compare with the direct analysis procedure. Elution is carried out by MALDI matrix solution to get them out of the entangled nanotubes, which are difficult to desorb by laser due to the complex nanotube structures. The advantage of these optimized methods compared to the conventional screening methods is the improved sensitivity, selectivity and the short analysis time without prior albumin and immunoglobulin depletion. The comparison of similarly modified diamond and carbon nanotubes exhibit differences in their nature to bind the proteins out of serum due to the differences in their physical characteristics. Infrared (IR) spectroscopy provided hint for the presence of tertiary amine peak at the crucial chemical step of iminodiacetic acid addition to acid chloride functionality on carbon nanotubes. Atomic absorption spectroscopy (AAS) was utilized to quantitatively measure the copper capacity of these derivatized carbon nanotubes which is a direct measure of capacity of

  6. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

    Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan;

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be...

  7. Serum protein profiles predict coronary artery disease in symptomatic patients referred for coronary angiography

    LaFramboise William A

    2012-12-01

    Full Text Available Abstract Background More than a million diagnostic cardiac catheterizations are performed annually in the US for evaluation of coronary artery anatomy and the presence of atherosclerosis. Nearly half of these patients have no significant coronary lesions or do not require mechanical or surgical revascularization. Consequently, the ability to rule out clinically significant coronary artery disease (CAD using low cost, low risk tests of serum biomarkers in even a small percentage of patients with normal coronary arteries could be highly beneficial. Methods Serum from 359 symptomatic subjects referred for catheterization was interrogated for proteins involved in atherogenesis, atherosclerosis, and plaque vulnerability. Coronary angiography classified 150 patients without flow-limiting CAD who did not require percutaneous intervention (PCI while 209 required coronary revascularization (stents, angioplasty, or coronary artery bypass graft surgery. Continuous variables were compared across the two patient groups for each analyte including calculation of false discovery rate (FDR ≤ 1% and Q value (P value for statistical significance adjusted to ≤ 0.01. Results Significant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100, C-reactive protein (CRP, fibrinogen, vascular cell adhesion molecule 1 (VCAM-1, myeloperoxidase (MPO, resistin, osteopontin, interleukin (IL-1β, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP and decreased apolipoprotein A1 (APO-A1. Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity while maintaining 95% sensitivity for patients requiring

  8. Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles

    Mokry, M.; Hatzis, P.; de Bruijn, E.; Koster, J.; Versteeg, R.; Schuijers, J.; van de Wetering, M.L.; Guryev, V.; Clevers, H.; Cuppen, E.

    2010-01-01

    Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be non-

  9. Stomatin-like protein 2 of turbot Scopthalmus maximus: Gene cloning, expression profiling and immunoregulatory properties.

    Chi, Heng; Hu, Yong-Hua

    2016-02-01

    Stomatin-like protein 2 (SLP-2) is a novel and unusual member of the stomatin gene superfamily. In this study, we obtained a full-length SLP-2 (SmSLP-2) cDNA from turbot (Scopthalmus maximus) spleen cDNA library. The cDNA sequence of SmSLP-2 contains a 5'-UTR of 107 bp, an ORF of 1050 bp, and a 3'-UTR of 959 bp. The ORF encodes a putative protein of 349 residues, which has a calculated molecular mass of 38.7 kDa. The SmSLP-2 protein possesses a prohibitin-homology (PHB) domain (residues 40 to 198) and shares 72.4-87.6% overall sequence identity with that of the teleost species. The highest expression of SmSLP-2 mRNA was found in the skin, followed by the head kidney, gut, spleen, liver, heart, gill and muscle. Moreover, both viral and bacterial pathogen infection resulted in the up-regulation of SmSLP-2 mRNA in the turbot head kidney and spleen in vivo. Subcellular localization analysis indicated that the SmSLP-2 proteins are mainly located in the peripheral membrane of ZF4 cells. This study also demonstrated that SmSLP-2 modulates IL-2 expression via active NFκB signaling pathway, and is possibly involved in host immune defense against bacterial and viral pathogens. PMID:26806162

  10. Special low protein foods for phenylketonuria : availability in Europe and an examination of their nutritional profile

    Pena, Maria Joao; Almeida, Manuela Ferreira; van Dam, Esther; Ahring, Kirsten; Belanger-Quintana, Amaya; Dokoupil, Katharina; Gokmen-Ozel, Hulya; Lammardo, Anna Maria; MacDonald, Anita; Robert, Martine; Rocha, Julio Cesar

    2015-01-01

    Background: Special low protein foods (SLPF) are essential in the nutritional management of patients with phenylketonuria (PKU). The study objectives were to: 1) identify the number of SLPF available for use in eight European countries and Turkey and 2) analyse the nutritional composition of SLPF av

  11. Intestinal microbes influence the survival, reproduction and protein profile of Trichinella spiralis in vitro.

    Jiang, Hai-yan; Zhao, Na; Zhang, Qiao-ling; Gao, Jiang-ming; Liu, Li-li; Wu, Teng-Fei; Wang, Ying; Huang, Qing-hua; Gou, Qiang; Chen, Wei; Gong, Peng-tao; Li, Jian-hua; Gao, Ying-jie; Liu, Bo; Zhang, Xi-chen

    2016-01-01

    The interactions between intestinal microbes and parasitic worms play an essential role in the development of the host immune system. However, the effects of gut microbes on Trichinella spiralis are unknown. The aim of this work was to explore microbe-induced alterations in the survival and reproduction of T. spiralis in vitro. To further identify the proteins and genes involved in the response of nematodes to microbes, quantitative proteomic analysis of T. spiralis was conducted by iTRAQ-coupled LCMS/MS technology and quantitative real-time-PCR was used to measure changes in mRNA expression. The results showed Lactobacillus acidophilus, and especially Lactobacillus bulgaricus, significantly enhanced the survival and reproductive rates of nematodes. Salmonella enterica, and especially Escherichia coli O157:H7 (EHEC), had opposite effects. Genetic responses were activated mainly by EHEC. A total of 514 proteins were identified and quantified, and carbohydrate metabolism-related proteins existed in a higher proportion. These findings indicated that some gut bacteria are friendly or harmful to humans and in addition they may have similar beneficial or detrimental effects on parasites. This may be due to the regulation of expression of specific genes and proteins. Our studies provide a basis for developing therapies against parasitic infections from knowledge generated by studying the gut microbes of mammals. PMID:26432293

  12. Vliv pivovarského procesu na profil proteinů ječmene

    Benkovská, Dagmar; Flodrová, Dana; Psota, V.; Bobálová, Janette

    2011-01-01

    Roč. 57, 7-8 (2011), 260-265. ISSN 0023-5830 R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : barley * proteins * brewing process Subject RIV: CB - Analytical Chemistry, Separation

  13. Identification of biomarkers for radiation-induced acute intestinal symptoms (RIAISs) in cervical cancer patients by serum protein profiling

    Radiation-induced acute intestinal symptoms (RIAISs) are the most frequent complication of radiotherapy that causes great pain and limits the treatment efficacy. The aim of this study was to identify serum biomarkers of RIAISs in cervical cancer patients by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Serum samples were collected from 66 cervical cancer patients prior to pelvic radiotherapy. In our study, RIAISs occurred in 11 patients. An additional 11 patients without RIAISs were selected as controls, whose age, stage, histological type and treatment methods were matched to RIAISs patients. The 22 sera were subsequently analyzed by SELDI-TOF MS, and the resulting protein profiles were evaluated to identify biomarkers using appropriate bioinformatics tools. Comparing the protein profiles of serum samples from the RIAIS group and the control group, it was found that 22 protein peaks were significantly different (P < 0.05), and six of these peaks with mass-to-charge (m/z) ratios of 7514.9, 4603.94, 6887.41, 2769.21, 3839.72 and 4215.7 were successfully identified. A decision tree model of biomarkers was constructed based on three biomarkers (m/z 1270.88, 1503.23 and 7514.90), which separated RIAIS-affected patients from the control group with an accuracy of 81%. This study suggests that serum proteomic analysis by SELDI-TOF MS can identify cervical cancer patients that are susceptible to RIAISs prior to pelvic radiotherapy. (author)

  14. Examination on the protein profiles of salivary glands of P. berghei infected anopheles Sp. post gamma irradiation using SDS-PAGE technique for developing malaria vaccine

    Sporozoite is a step of malaria parasitic live cycle that is most invasive and appropriate vaccine candidate. Result of experiments showed that malaria vaccine created by attenuating Plasmodium sp sporozoites with gamma rays was proven more effective. Study on the effects of irradiation to the profiles of protein in vaccine development is also important. The aim of this research was to examine the protein profile of salivary glands in sporozoite infected Anopheles sp post gamma irradiation using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. Examination covered the infection of Anopheles sp with Plasmodium sp, maintenance of infected mosquitoes for 14-16 days to obtain sporozoites, in vivo - in vitro irradiation of mosquitoes, preparation of salivary glands, electrophoresis on 10% SDS-PAGE, and Commassie blue staining. Results showed a different protein profile of infected and non infected salivary glands of Anopheles sp. There was additional protein band numbers at higher dose of irradiation (200 Gy) from sporozoite protein of P. berghei (MW 62 kDa). However, no difference of the profiles of circumsporozoite protein (CSP) observed among gamma irradiation doses of 150, 175 and 200 Gy. These results provide basic information that would lead to further study on the role of sporozoite proteins in malaria vaccine development. (author)

  15. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

    D Malfacini

    Full Text Available Nociceptin/orphanin FQ (N/OFQ controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP. Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells. In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

  16. Multiplex protein profiling of bronchoalveolar lavage in idiopathic pulmonary fibrosis and hypersensitivity pneumonitis

    Willems Stijn

    2013-01-01

    Full Text Available Context: Idiopathic pulmonary fibrosis (IPF and chronic hypersensitivity pneumonitis (HP are diffuse parenchymal lung diseases characterized by a mixture of inflammation and fibrosis, leading to lung destruction and finally death. AIMS: The aim of this study was to compare different pathophysiological mechanisms, such as angiogenesis, coagulation, fibrosis, tissue repair, inflammation, epithelial damage, oxidative stress, and matrix remodeling, in both disorders using bronchoalveolar lavage (BAL. Methods: At diagnosis, patients underwent bronchoscopy with BAL and were divided into three groups: Control ( n = 10, HP ( n = 11, and IPF ( n = 11, based on multidisciplinary approach (clinical examination, radiology, and histology: Multiplex searchlight technology was used to analyze 25 proteins representative for different pathophysiological processes: Eotaxin, basic fibroblast growth factor (FGFb, fibronectin, hepatocyte growth factor (HGF, interleukine (IL-8, IL-12p40, IL-17, IL-23, monocyte chemotactic protein (MCP-1, macrophage-derived chemokine (MDC, myeloperoxidase (MPO, matrix metalloproteinase (MMP-8, MMP-9, active plasminogen activating inhibitor 1 (PAI-1, pulmonary activation regulated chemokine (PARC, placental growth factor (PlGF, protein-C, receptor for advanced glycation end products (RAGE, regulated on activation normal T cells expressed and secreted (RANTES, surfactant protein-C (SP-C, transforming growth factor-β1 (TGF-β1, tissue inhibitor of metalloproteinase-1 (TIMP-1, tissue factor, thymic stromal lymphopoietin (TSLP, and vascular endothelial growth factor (VEGF. Results: All patients suffered from decreased pulmonary function and abnormal BAL cell differential compared with control. Protein levels were increased in both IPF and HP for MMP-8 ( P = 0.022, MMP-9 ( P = 0.0020, MCP-1 ( P = 0.0006, MDC ( P = 0.0048, IL-8 ( P = 0.013, MPO ( P = 0.019, and protein-C ( P = 0.0087, whereas VEGF was decreased ( P = 0.0003 compared with

  17. Amino acids fortification of low-protein diet for broilers under tropical climate: ideal essential amino acids profile

    Elmutaz Atta Awad

    2014-05-01

    Full Text Available A three-week trial was conducted to determine the effect of lowering dietary protein level (DPL with optimal amino acid (AA profile on growth performance, blood metabolites, and relative weights of abdominal fat and internal organs in broiler chickens raised under tropical hot and humid environment. Five isocaloric (3023 metabolisable energy/kg starter (1-21 days experimental diets were formulated in a gradual crude protein (CP decline from 22.2 (control to 16.2% by 1.5% interval. All diets were meeting or exceeding National Research Council recommendations except CP and metabolisable energy. The formulations were also adjusted to contain 1.1 digestible Lys to meet the ideal AA ratios concept. Body weights (BW, weight gains (WG, feed intake and feed conversion ratio of groups with 19.2, 20.7 and 22.2% DPL were not significantly different. However, BW and WG suppressed (P<0.05 with 16.2 and 17.7% DPL. Feeding the 16.2% CP diet significantly reduced serum total protein and uric acid, but increased serum triglyceride (P<0.05. Moreover, relative heart weights increased (P<0.05 but no changes occurred in liver and abdominal fat weights in chicks with 16.2% DPL. In summary, CP of broilers starter (1-21 days diet can be reduced till 19.2% with essential AA fortification and without any adverse effect on growth performance under the hot, humid tropics.

  18. Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis

    Jarred W. Rensvold

    2013-01-01

    Full Text Available Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.

  19. Expression Profiling of Human Genetic and Protein Interaction Networks in Type 1 Diabetes

    Brunak, Søren; Bergholdt, R; Brorsson, C;

    2009-01-01

    : oxidative stress, regulation of transcription and apoptosis. To understand biological systems, integration of genetic and functional information is necessary, and the current study has used this approach to improve understanding of T1D and the underlying biological mechanisms.......Proteins contributing to a complex disease are often members of the same functional pathways. Elucidation of such pathways may provide increased knowledge about functional mechanisms underlying disease. By combining genetic interactions in Type 1 Diabetes (T1D) with protein interaction data we have...... previously identified sets of genes, likely to represent distinct cellular pathways involved in T1D risk. Here we evaluate the candidate genes involved in these putative interaction networks not only at the single gene level, but also in the context of the networks of which they form an integral part. m...

  20. A Gram-Negative Bacterial Secreted Protein Types Prediction Method Based on PSI-BLAST Profile

    2016-01-01

    Prediction of secreted protein types based solely on sequence data remains to be a challenging problem. In this study, we extract the long-range correlation information and linear correlation information from position-specific score matrix (PSSM). A total of 6800 features are extracted at 17 different gaps; then, 309 features are selected by a filter feature selection method based on the training set. To verify the performance of our method, jackknife and independent dataset tests are performed on the test set and the reported overall accuracies are 93.60% and 100%, respectively. Comparison of our results with the existing method shows that our method provides the favorable performance for secreted protein type prediction.

  1. Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

    Nourizadeh-Lillabadi, Rasoul; Seilø Torgersen, Jacob; Vestrheim, Olav; König, Melanie; Aleström, Peter; Syed, Mohasina

    2010-01-01

    Background The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed ...

  2. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Bjorn; Moritz, Thomas; Hvidsten, Torgeir R.; Trygg, Johan; Wingsle, Gunnar

    2016-01-01

    Background: Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and var...

  3. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice

    Yi-Ming Chen; Che-Li Lin; Li Wei; Yi-Ju Hsu; Kuan-Neng Chen; Chi-Chang Huang; Chin-Hsung Kao

    2016-01-01

    Exercise and fitness training programs have attracted the public’s attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. Purpose: In this study, we designed a power exercise training (PET) program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA)-rich sake protein (SP) to determine whether the supplement had a syne...

  4. Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

    Dernick, Gregor; Obermüller, Stefan; Mangold, Cyrill; Magg, Christine; Matile, Hugues; Gutmann, Oliver; von der Mark, Elisabeth; Handschin, Corinne; Maugeais, Cyrille; Niesor, Eric J.

    2011-01-01

    The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipo...

  5. Special low protein foods for phenylketonuria: availability in Europe and an examination of their nutritional profile

    Pena, Maria Joao; Almeida, Manuela Ferreira; van Dam, Esther; Ahring, Kirsten; Belanger-Quintana, Amaya; Dokoupil, Katharina; Gokmen-Ozel, Hulya; Lammardo, Anna Maria; MacDonald, Anita; Robert, Martine; Rocha, Julio Cesar

    2015-01-01

    Background: Special low protein foods (SLPF) are essential in the nutritional management of patients with phenylketonuria (PKU). The study objectives were to: 1) identify the number of SLPF available for use in eight European countries and Turkey and 2) analyse the nutritional composition of SLPF available in one of these countries. Methods: European Nutritionist Expert Panel on PKU (ENEP) members (Portugal, Spain, Belgium, Italy, Germany, Netherlands, UK, Denmark and Turkey) provided data on...

  6. Special low protein foods for phenylketonuria: availability in Europe and an examination of their nutritional profile

    Pena, M.; Almeida, M.; van Dam, E; Ahring, K.; Bélanger-Quintana, A.; K. Dokoupil; H. Gokmen-Ozel; Lammardo, A.; MacDonald, A.; Robert, M.; Rocha, J

    2015-01-01

    Background Special low protein foods (SLPF) are essential in the nutritional management of patients with phenylketonuria (PKU). The study objectives were to: 1) identify the number of SLPF available for use in eight European countries and Turkey and 2) analyse the nutritional composition of SLPF available in one of these countries. Methods European Nutritionist Expert Panel on PKU (ENEP) members (Portugal, Spain, Belgium, Italy, Germany, Netherlands, UK, Denmark and Turkey) provide...

  7. Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies

    Seo, Young Ho; Carroll, Kate S.

    2009-01-01

    Hydrogen peroxide (H2O2) functions as a second messenger that can activate cell proliferation through chemoselective oxidation of cysteine residues in signaling proteins. The connection between H2O2 signaling, thiol oxidation, and activation of growth pathways has emerged as fertile ground for the development of strategies for cancer treatment. Central to achieving this goal is the development of tools and assays that facilitate characterization of the molecular events associated with tumorig...

  8. Special low protein foods for phenylketonuria: availability in Europe and an examination of their nutritional profile

    Pena, Maria João; Almeida, Manuela Ferreira; van Dam, Esther; Ahring, Kirsten; Bélanger-Quintana, Amaya; Dokoupil, Katharina; Gokmen-Ozel, Hulya; Lammardo, Anna Maria; MacDonald, Anita; Robert, Martine; Rocha, Júlio César

    2015-01-01

    Background Special low protein foods (SLPF) are essential in the nutritional management of patients with phenylketonuria (PKU). The study objectives were to: 1) identify the number of SLPF available for use in eight European countries and Turkey and 2) analyse the nutritional composition of SLPF available in one of these countries. Methods European Nutritionist Expert Panel on PKU (ENEP) members (Portugal, Spain, Belgium, Italy, Germany, Netherlands, UK, Denmark and Turkey) provided data on S...

  9. Different Polar Metabolites and Protein Profiles between High- and Low-Quality Japanese Ginjo Sake.

    Kei Takahashi

    Full Text Available Japanese ginjo sake is a premium refined sake characterized by a pleasant fruity apple-like flavor and a sophisticated taste. Because of technical difficulties inherent in brewing ginjo sake, off-flavors sometimes occur. However, the metabolites responsible for off-flavors as well as those present or absent in higher quality ginjo sake remain uncertain. Here, the relationship between 202 polar chemical compounds in sake identified using capillary electrophoresis coupled with time-of-flight mass spectrometry and its organoleptic properties, such as quality and off-flavor, was examined. First, we found that some off-flavored sakes contained higher total amounts of metabolites than other sake samples. The results also identified that levels of 2-oxoglutaric acid and fumaric acid, metabolites in the tricarboxylic acid cycle, were highly but oppositely correlated with ginjo sake quality. Similarly, pyridoxine and pyridoxamine, co-enzymes for amino transferase, were also highly but oppositely correlated with ginjo sake quality. Additionally, pyruvic acid levels were associated with good quality as well. Compounds involved in the methionine salvage cycle, oxidative glutathione derivatives, and amino acid catabolites were correlated with low quality. Among off-flavors, an inharmonious bitter taste appeared attributable to polyamines. Furthermore, protein analysis displayed that a diversity of protein components and yeast protein (triosephosphate isomerase, TPI leakage was linked to the overall metabolite intensity in ginjo sake. This research provides insight into the relationship between sake components and organoleptic properties.

  10. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

  11. SILAC based protein profiling data of MKK3 knockout mouse embryonic fibroblasts.

    Srivastava, Anup; Shinn, Amanda S; Lam, TuKiet T; Lee, Patty J; Mannam, Praveen

    2016-06-01

    This data article reports changes in the phospho and total proteome of MKK3 knock out (MKK3(-) (/) (-)) mouse embryonic fibroblasts (MEFs). The dataset generated highlights the changes at protein level which can be helpful for understanding targets of the MAP kinase signaling pathway. Data was collected after TiO2-based phosphopeptide enrichment of whole cell lysate at baseline condition with bottom-up SILAC-based LC MS/MS quantitative mass spectrometry. We report all the proteins and peptides identified and quantified in MKK3(-/-) and WT MEFs. The altered pathways in MKK3(-/-) MEFs were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) and Ingenuity Pathway Analysis (IPA) and are presented as a table and graph, respectively. The data reported here is related to the published work [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs. PMID:26977448

  12. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  13. Amino acid profile of raw and locally processed seeds of Prosopis africana and Ricinus communis: potential antidotes to protein malnutrition

    Chidi U. Igwe

    2012-04-01

    Full Text Available Background: Increasing incidence of malnutrition occasioned by high incidence of hunger,worsening food situation in the world, insufficient availability and high cost of animal protein sources, has necessitated extensive research into and use of alternative plant protein sources especially underexploited leguminous seeds.Methods: Flours from raw, boiled and fermented seeds of Prosopis africana and Ricinus communis were evaluated for crude protein and amino acid (AA profiles, and their protein qualities determined. Results: Fermentation improved the protein contents of raw seeds of P. africana and R. communis by 18.70% and 3.95% respectively. In the raw and fermented P. africana seeds, glutamate at 132.60 ± 1.30 and 182.70 ± 3.02 mg/g crude protein (mg/gcp was the most abundant amino acid (AA, while leucine (62.80 ± 0.60 and 79.50 ± 2.01 mg/gcp was the most concentrated essential amino acid (EAA. Aspartate (151.90 ± 2.01 and 170.10 ± 2.00 mg/gcp and arginine (72.80 ± 2.01 and 78.60 ± 2.00 mg/gcp were the most concentrated and abundant non-essential amino acid (NEAA and EAA in the raw and fermented samples of R. communisrespectively. The total AA concentrations (mg/gcp of raw and fermented P. africana were 733.00 and 962.60 respectively, while those of R. communis were 823.50 and 894.10 respectively. The total EAA contents (mg/gcp for P. africana were 311.00 (raw and 404.50 (fermented, and for R. communis; 401.10 (raw and 430.30 (fermented. Threonine was the limiting EAA in raw and fermented P. africana, whereas lysine was the limiting EAA in R. communis raw sample. Fermentation significantly (p<0.05 increased the individual AA compositions of P. africana and R. communis by 94% and 53% respectively, while boiling reduced these parameters significantly (p<0.05 by 47% and 82% respectively. Conclusion: P. africana and R. communis seeds are potentially important plant sources of protein and essential amino acids, and so could be of great

  14. Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

    Bieghs, Liesbeth; Brohus, Malene; Kristensen, Ida B;

    2016-01-01

    and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM.......6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the...... profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration....

  15. Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

    Taruna Khurana

    Full Text Available German cockroach (GCr allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.Single chain fragment variable (scFv antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.Chicken scFv's generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv's recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

  16. Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

    Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS). Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the 'GenePattern' suite of computational tools (Broad Institute, MIT, USA). Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR) test statistic was used to identify and rank differentially expressed marker peaks. The k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, 'leave-one-out' cross-validation method. 73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour verses adjacent normal mucosa tissue (P ≤ 0.01, false discovery rate ≤ 0.05). Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100% sensitivity and specificity (95% confidence interval: 67.9-99.2%). Supervised prediction using 'leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours (P = < 0.001; receiver-operator characteristics - ROC - error, 0.171); disease recurrence was correctly predicted in 5/6 cases and disease-free survival (median follow-up time, 25 months) was correctly predicted in 22

  17. Influence of age on profile of serum proteins in the calf

    Piccione Giuseppe; Casella Stefania; Giannetto Claudia; Vazzana Irene; Niutta P.P.; Giudice Elisabetta

    2009-01-01

    The authors studied the course of serum proteins during the first week and the first month of life in the calf in order to obtain useful information for neonatal care. Eight Limousine calves, four males and four females, clinically healthy, were used. From all animals blood samples were collected from the external jugular vein from the day after the birth every day for one week and every five days for thirty days. Blood samples were taken at the same hour (09:00), and the serum concentrations...

  18. Seasonal influence on biochemical profile and serum protein electrophoresis for Boa constrictor amarali in captivity.

    Silva, L F N; Riani-Costa, C C M; Ramos, P R R; Takahira, R K

    2011-05-01

    Similarly to other reptiles, snakes are ectothermic animals and depend exclusively on the environment for the maintenance of their physiological, biochemical and immunological processes. Thus, changes in biochemical values can be expected due to seasonal influence. Twenty-two adult specimens of Boa constrictor amarali kept in captivity were used. Blood collections were done in two different seasons: winter (July 2004) and summer (January 2005) for the following assays: uric acid, aspartate aminotransferase (AST), glucose, cholesterol, total protein, and serum protein electrophoresis. The mean biochemical results found in summer and winter, respectively, were: 6.3 ± 3.4 and 11.3 ± 6.2 mg/dL for uric acid; 28.7 ± 12.4 and 20.7 ± 16.2 UI/L for AST; 26.3 ± 17 and 17.4 ± 6.8 mg/dL for glucose; 67.3 ± 30.2 and 69.7 ± 38.5 mg/dL for cholesterol; and 5.9 ± 1.6 and 5.9 ± 1.4 g/dL for total protein. Results regarding electrophoresis in summer and winter, respectively, were: 1.9 ± 0.7 and 2.4 ± 0.6 g/dL for albumin; 0.7 ± 0.2 and 0.5 ± 0.2 g/dL for α-globulin; 1.5 ± 0.5 and 1.7 ± 0.6 g/dL for β-globulin; and 1.8 ± 0.5 and 1.5 ± 0.5 g/dL for γ-globulin. In the summer, there was a significant increase in AST and a decrease in uric acid (p < 0.05). Serum protein electrophoresis showed a significant increase in α-globulin fraction (p < 0.05) in the same season. There were not significant differences between seasons for the remaining variables. Based on these results, the period of the year must be considered in the interpretation of some biochemical values for these animals. PMID:21755171

  19. Study on Outer Membrane Protein (OMP Profile of Aeromonas Strains using SDS- PAGE

    N Sachan

    2012-06-01

    Full Text Available Mesophilic aeromonads are being increasingly reported pathogen of humans and lower vertebrates. Water and foods are considered to be the chief source of Aeromonas spp. At present there are several techniques available for the detection of Aeromonas spp. from water and foods. However, there is still need to develop immunodiagnostics for rapid detection of Aeromonas spp. irrespective of their species or serotype. To meet out this requirement present study was undertaken to identify the common protein moiety in their OMPs by SDS-PAGE so, that immunoassays can be developed for efficient and rapid detection of Aeromonas spp. from foods. [Vet. World 2012; 5(3.000: 173-177

  20. Effects of stem cell therapy on protein profile of parkinsonian rats using an(18) O-labeling quantitative proteomic approach.

    Liu, Yahui; Liu, Kefu; Qin, Wei; Liu, Chenghao; Zheng, Xiaowei; Deng, Yulin; Qing, Hong

    2016-03-01

    The application of neural stem cell (NSC) research to neurodegenerative diseases has led to promising clinical trials. Currently, NSC therapy is most promising for Parkinson's disease (PD). We conducted behavioral tests and immunoassays for the profiling of a PD model in rats to assess the therapeutic effects of NSC treatments. Further, using a multiple sample comparison workflow, combined with (18) O-labeled proteome mixtures, we compared the differentially expressed proteins from control, PD, and NSC-treated PD rats. The results were analyzed bioinformatically and verified by Western blot. Based on our initial findings, we believe that the proteomic approach is a valuable tool in evaluating the therapeutic effects of NSC transplantation on neurodegenerative disorders. PMID:26791447

  1. Changes in the salivary protein profile of morbidly obese women either previously subjected to bariatric surgery or not.

    Lamy, Elsa; Simões, Carla; Rodrigues, Lénia; Costa, Ana Rodrigues; Vitorino, Rui; Amado, Francisco; Antunes, Célia; do Carmo, Isabel

    2015-12-01

    Saliva is a non-invasive source of biomarkers useful in the study of physiological mechanisms. Moreover, this fluid has diverse functions, among which food perception and ingestion, making it particularly suitable for the study of obesity. The aims of this study were to assess changes in salivary proteome among morbidly obese women, with a view to provide information about mechanisms potentially related to the development of obesity, and to evaluate whether these changes persist after weight loss. Mixed saliva samples from morbidly obese women (N = 18) who had been either subjected (group O-BS) or not (group O) to bariatric surgery and women with normal weight (N = 14; group C) were compared for protein profiles, alpha-amylase abundance and enzymatic activity, and carbonic anhydrase (CA) VI abundance. Differences in salivary obese profiles were observed for 23 different spots. Zinc-alpha-2 glycoprotein-containing spots showed higher abundance in group O only, whereas cystatin S-containing spots presented higher abundance in the two groups of obese subjects. Most of the spots identified as salivary amylase were present at lower levels in group O-BS. With regard to the amylase enzymatic activity, increases were observed for group O and decreases for group O-BS. One interesting finding was the high correlation between levels of CA VI and body mass index in group O, which was not observed for groups O-BS or C. The differences between groups, mainly regarding salivary proteins involved in taste sensitivity and metabolism, point to the potential of using saliva in the study of obesity development. PMID:26399515

  2. Multiplexed profiling of secreted proteins for the detection of potential space biomarkers.

    Dieriks, Birger; De Vos, Winnok H; Moreels, Marjan; Ghardi, Myriam; Hennekam, Raoul; Broers, Jos L V; Baatout, Sarah; van Oostveldt, Patrick

    2011-01-01

    Space travel exposes astronauts to a plethora of potentially detrimental conditions, such as cosmic radiation and microgravity. As both factors are hard to simulate on Earth, present knowledge remains limited. However, this knowledge is of vital importance, making space flight experiments a necessity for determining the biological effects and the underlying biochemical processes, especially when keeping future long-term interplanetary missions in mind. Instead of estimating the long-term effects, which usually implicate severe endpoints (e.g., cancer) and which are often difficult to attribute, research has shifted to finding representative biomarkers for rapid and sensitive detection of individual radiosensitivity. In this context, an appealing set of candidate markers is the group of secreted proteins, as they exert an intercellular signaling function and are easy to assess. We screened a subset of secreted proteins in cells exposed to space travel by means of multiplex bead array analysis. To determine the cell-specific signatures of the secreted molecules, we compared the conditioned medium of normal fibroblast cells to fibroblasts isolated from a patient with Hutchinson-Gilford Progeria syndrome, which are known to have a perturbed nuclear architecture and DNA damage response. Out of the 88 molecules screened, 20 showed a significant level increase or decrease, with a differential response to space conditions between the two cell types. Among the molecules that were retained, which may prove to be valuable biomarkers, are apolipoprotein C-III, plasminogen activator inhibitor type 1, β-2-microglobulin, ferritin, MMP-3, TIMP-1 and VEGF. PMID:21461557

  3. Relationship of molecular weight distribution profile of unreduced gluten protein extracts with quality characteristics of bread.

    Chaudhary, Nisha; Dangi, Priya; Khatkar, B S

    2016-11-01

    A statistical correlation was established among the molecular weight distribution patterns of unreduced gluten proteins and physicochemical, rheological and bread-making quality characteristics of wheat varieties. Size exclusion chromatography fractionated the gluten proteins apparently into five peaks. Peak I signified glutenins (30-130kDa), peak II as gliadins (20-55kDa), peak III as very low molecular weight monomeric gliadins (10-28kDa), peak IV and V, collectively, as albumins and globulins (gluten index (r=0.959(∗∗)), and gliadin/glutenin ratio (r=-0.952(∗∗)), while peak II influenced inversely as expected. Peak I exhibited positive statistical significance with bread loaf volume (r=0.848(∗∗)); however, peak II had negative (r=-0.818(∗∗)) impact. Bread firmness increased with increment in peak II (r=0.625(∗∗)), and decreased with accretion in peak I (r=-0.623(∗∗)). PMID:27211654

  4. The Characterization of Protein Profiles of the Aeromonas hydrophila and A. caviae Strains Isolated from Gull and Rainbow Trout Feces by SDS-PAGE

    KÖRKOCA, Hanifi; BOYNUKARA, Banur

    2003-01-01

    Ten motile Aeromonads, including one Aeromonas hydrophila ATCC 7512, were used. Of the strains, five were isolated from gulls (one A. hydrophila strain and four A. caviae strains) and four from rainbow trout (three A. hydrophila strains and one A. caviae strain). Whole cell protein profiles of these strains were analyzed by SDS-PAGE. Forty-five pairs were formed in comparing the protein profiles of each strain. Of the pairs, 17 (37.8%) were different, 18 (40%) were unclear and 10 (22.2%) were...

  5. Expression profile and protein translation of TMEM16A in murine smooth muscle

    Davis, Alison J; Forrest, Abigail S; Jepps, Thomas Andrew;

    2010-01-01

    , and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent......(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific...... for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle...

  6. Quality traits analysis and protein profiling of field pea (Pisum sativum) germplasm from Himalayan region.

    Sharma, Shagun; Singh, Narpinder; Virdi, Amardeep Singh; Rana, Jai Chand

    2015-04-01

    The grain and flour characteristics of different field pea (FP) accessions were evaluated. Accessions with higher grain weight had less compact structure with a greater proportion of large-sized starch granules. Accessions with higher protein content had lower starch content, blue value and λ(max) whereas accessions with higher amylose showed higher resistant starch (RS) and final viscosity and lower rapidly digestible starch (RDS). Ca, Zn, K and Fe content vary significantly amongst different accessions and creamish green and white seeds accessions showed higher Fe and Zn content. Yellow coloured accessions (1.36-3.71%) showed lower antioxidant activity as compared to brownish and green coloured accessions (4.06-9.30%). Out of 21 major polypeptides observed (9-100 kDa), 11 showed differential trypsin inhibitory activity (TIA) under non-reducing conditions. Polypeptides of 68, 46, 33 and 22 kDa showed prominent TIA. PMID:25442588

  7. Kafirin Protein Based Electrospun Fibers with Tunable Mechanical Property, Wettability, and Release Profile.

    Xiao, Jie; Shi, Ce; Zheng, Huijuan; Shi, Zhen; Jiang, Dong; Li, Yunqi; Huang, Qingrong

    2016-04-27

    Kafirin (KAF), the prolamine protein from sorghum grain, is a promising resource for fabricating renewable and biodegradable materials. However, research efforts in fulfilling its potentials are still lacking. In this work, electrospun kafirin fibers from acetic acid/dichloromethane solutions are reported for the first time. Biodegradable polycaprolactone (PCL) was blended with kafirin to obtain hybrid KAF/PCL fiber mats with desirable physical properties. Hydrogen bonding between the N-H group of kafirin and the C═O group of PCL was detected in each blended formulation. Our small-angle X-ray scattering results indicated that the long spacing decreased and the average spacing between crystalline lamellae of PCL increased with the increase of kafirin content. Compared to the hydrophobic surface of neat PCL fiber mat, KAF/PCL fiber mats under most of the blend ratios showed hydrophilic surface character, and the swelling property was composition-dependent. The fiber mats evolved from brittle ones to flexible ones with the increase of relative content of PCL. The most desirable mechanical performance was obtained at a kafirin/PCL mass blend ratio of 1:2. To simulate the nutraceutical release in body fluid, carnosic acid (CA) was selected as a nutraceutical model, and release behaviors in selected KAF/PCL fiber mats were found to be diffusion controlled. Whereas the amorphous region of kafirin dominated the release rate, PCL functioned as a hydrophobic skeleton to maintain the 3D scaffold of the fiber matrix. The fabricated KAF/PCL fiber mats open up new applications of underutilized cereal protein in nutraceutical delivery. PMID:27032442

  8. Expression Profile and Tissue-Specific Distribution of the Receptor-Interacting Protein 3 in BALB/c Mice.

    Wang, Qingnan; Yu, Meng; Zhang, Kaizhao; Liu, Jianxin; Tao, Pan; Ge, Shikun; Ning, Zhangyong

    2016-08-01

    RIP3, a member of receptor-interacting protein family, is serine/threonine kinase that contributes to necrosis and promotes systematic inflammation. However, detailed information of the expression pattern and tissue distribution in BALB/c mice, a commonly used laboratory animal model, is still unavailable. Here, we provided the basic data of expression profile and histologic distribution of RIP3 in tissues of BALB/c mice. Rip3 mRNA expression levels and tissue distribution were detected by real-time quantitative PCR and immunohistochemical detection, respectively. Rip3 mRNA expression showed the highest level in the spleen and duodenum, while with the lowest level in brain. Immunohistochemical detection revealed this protein located in different type cells in different tissues. What's more, the obvious positive staining in nuclear was detected in liver cells and neurons in cerebral cortex of the brain, while cells in other organs, including heart, spleen, lung, kidney, stomach, duodenum and trachea, showed strong positive mainly in cytoplasm. The results will help us to further understand the site-specific functions of RIP3 in necrosis and inflammatory responses. PMID:26969469

  9. Low-flow sheathless capillary electrophoresis-mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins.

    Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W

    2013-02-19

    Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars. PMID:23323765

  10. Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

    Wissenbach, Dirk K.; Pfeiffer, Susanne E. M.; Baumann, Sven; Hofbauer, Lorenz C.; von Bergen, Martin; Kalkhof, Stefan; Rammelt, Stefan

    2016-01-01

    Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis. PMID:27441377

  11. Exopolysaccharide-associated protein sorting in environmental organisms: the PEP-CTERM/EpsH system. Application of a novel phylogenetic profiling heuristic

    Ward Naomi

    2006-08-01

    Full Text Available Abstract Background Protein translocation to the proper cellular destination may be guided by various classes of sorting signals recognizable in the primary sequence. Detection in some genomes, but not others, may reveal sorting system components by comparison of the phylogenetic profile of the class of sorting signal to that of various protein families. Results We describe a short C-terminal homology domain, sporadically distributed in bacteria, with several key characteristics of protein sorting signals. The domain includes a near-invariant motif Pro-Glu-Pro (PEP. This possible recognition or processing site is followed by a predicted transmembrane helix and a cluster rich in basic amino acids. We designate this domain PEP-CTERM. It tends to occur multiple times in a genome if it occurs at all, with a median count of eight instances; Verrucomicrobium spinosum has sixty-five. PEP-CTERM-containing proteins generally contain an N-terminal signal peptide and exhibit high diversity and little homology to known proteins. All bacteria with PEP-CTERM have both an outer membrane and exopolysaccharide (EPS production genes. By a simple heuristic for screening phylogenetic profiles in the absence of pre-formed protein families, we discovered that a homolog of the membrane protein EpsH (exopolysaccharide locus protein H occurs in a species when PEP-CTERM domains are found. The EpsH family contains invariant residues consistent with a transpeptidase function. Most PEP-CTERM proteins are encoded by single-gene operons preceded by large intergenic regions. In the Proteobacteria, most of these upstream regions share a DNA sequence, a probable cis-regulatory site that contains a sigma-54 binding motif. The phylogenetic profile for this DNA sequence exactly matches that of three proteins: a sigma-54-interacting response regulator (PrsR, a transmembrane histidine kinase (PrsK, and a TPR protein (PrsT. Conclusion These findings are consistent with the hypothesis

  12. Expression profiles of prion and doppel proteins and of their receptors in mouse splenocytes.

    Cordier-Dirikoc, Sevda; Zsürger, Nicole; Cazareth, Julie; Ménard, Baptiste; Chabry, Joëlle

    2008-08-01

    Doppel (Dpl) shares common structural features with the prion protein (PrP) whose pathologic isoform is considered as the causative agent of prion diseases. Although their physiological functions in the immune system remain largely unknown, we demonstrated that substantial amounts of PrP and Dpl are expressed by spleen cells notably B lymphocytes, granulocytes and DC, but not T lymphocytes and NK. To characterize trans-interacting partners of PrP and Dpl on mouse splenocytes, fluorescent PrP and Dpl tetramers were produced and used as tracers. Both tetramers specifically bind to B lymphocytes, dendritic cells, macrophages and granulocytes and in a lesser extend to T lymphocytes. No binding was observed on NK, follicular dendritic cells and mesenchymal spleen cells. The activation of intracellular transduction signals (i.e. intracellular calcium concentration and activation of the MAP kinase pathway) suggested that PrP and Dpl tetramers bind to functional receptors on B cells. None of the previously described PrP partners account for the binding sites characterized here. Our study suggests a possible role for PrP and Dpl in the cell-cell interactions in the immune system. PMID:18604867

  13. Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence.

    Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

    2016-07-01

    Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

  14. Leptin treatment in activity-based anorexia

    Hillebrand, Jacquelien J G; Koeners, Maarten P; de Rijke, Corine E; Kas, Martien J H; Adan, Roger A H

    2005-01-01

    BACKGROUND: Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa (AN). In ABA, scheduled feeding together with voluntary access to a running wheel results in increased running wheel activity (RWA), hypophagia, and body weight loss. Previously it was shown that leptin treat

  15. Plasma electrophoretic profiles and hemoglobin binding protein reference intervals in the eastern box turtle (Terrapene carolina carolina) and influences of age, sex, season, and location.

    Flower, Jennifer E; Byrd, John; Cray, Carolyn; Allender, Matthew C

    2014-12-01

    Evaluation of plasma electrophoretic profiles and acute phase protein concentrations may play a valuable role in health assessment of reptiles; however, little is known about reference intervals in free-ranging eastern box turtles (Terrapene carolina carolina). The purpose of this study was to establish reference intervals of protein electrophoretic profiles and hemoglobin binding protein ([HBP] as determined by a haptoglobin assay) in free-ranging eastern box turtles and to assess any possible correlations between varying age class (adults vs. juvenile), sex (male, female, or unknown), season (spring, summer, or fall), or location (Tennessee vs. Illinois). Blood samples were obtained from 324 eastern box turtles from 2010 to 2012 at three sites in Illinois and one site in Tennessee, USA. Significant differences were observed with total protein (sex, season, state, Illinois location), albumin (age class, season, state, Illinois location), α-1 globulins (sex, season, Illinois location), α-2 globulins (sex, season, state, Illinois location), β globulins (age class, sex, season, state, Illinois location), γ globulins (sex, season state, Illinois location), and hemoglobin binding protein (age class, sex, state, Illinois location). The use of electrophoretic profiles and acute phase proteins is a relatively new concept in reptilian medicine, and this study allowed for establishment of references intervals in the eastern box turtle and emphasized differences that occured based on age, sex, season, and location. Future research in this area can now build on these data to determine changes in population health over time or alterations due to specific environmental or disease threats. PMID:25632671

  16. Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens

    Islam, Md. Zaherul; Yun, Hae Keun

    2016-01-01

    Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585), VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132) showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674) were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi) after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines. PMID:27493610

  17. Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer

    Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series

  18. Expression profiles of inhibitor of growth protein 2 in normal and cancer tissues: An immunohistochemical screening analysis.

    Zhao, Shuang; Yang, Xue-Feng; Gou, Wen-Feng; Lu, Hang; Li, Hua; Zhu, Zhi-Tu; Sun, Hong-Zhi; Zheng, Hua-Chuan

    2016-02-01

    Inhibitor of growth protein 2 (ING2) has an important role in the regulation of chromatin remodeling, cell proliferation, cell‑cycle arrest, senescence and apoptosis. The present study performed an immunohistochemical analysis for expression profiling of ING2 protein in an array of tissues comprising normal mouse and human tissues, as well as human hepatocellular (n=62), renal clear cell (n=62), pancreatic (n=62), esophageal squamous cell (n=45), cervical squamous cell (n=31), breast (n=144), gastric (n=196), colorectal (n=96), ovarian (n=208), endometrial (n=96) and lung (n=192) carcinoma tissues. In mouse tissues, ING2 was detected in the nuclei and cytoplasm of the glandular epithelium of breast, hepatocytes, intestine, bronchium and alveoli, as well as the squamous epithelium of skin and glomeruli, and in myocardial cells, while it was located in the cytoplasm of renal tubules and striated muscle cells. ING2 protein was scattered in the brain and spleen. In human tissues, ING2 protein was principally distributed in the cytoplasm, while in it was present in the cytoplasm and nuclei in the stomach, intestine, cervix, endometrium trachea, breast and pancreas. The nuclear location of ING2 in the stomach was more prominent than that in the cytoplasm. High ING2 immunoreactivity was detected in the tongue, stomach, skin, pancreas, cervix and breast, whereas weakly in the brain stem, thymus, thyroid, lung, striated muscle, testis, bladder and ovary. In total, 617 out of 1,194 of the tested cancer tissues (51.7%) were ING2-positive. In most cases, ING2 expression was found to be restricted to the cytoplasm of all cancer tissues, while in certain cancer types, including renal clear cell, ovarian and colorectal carcinoma, it was occasionally present in the nuclei. Among the cancer tissues examined, ING2 was most frequently expressed in breast cancer (67.4%) and gynecological cancer types, including ovarian cancer (61.5%) and endometrial cancer (57.3%). Compared with

  19. Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition

    Hansen, Mette; Lange, Marianne; Friis, Carsten;

    2007-01-01

    Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need to be...

  20. Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle

    Chang-Feng Chi; Fa-Yuan Hu; Bin Wang; Zhong-Rui Li; Hong-Yu Luo

    2015-01-01

    Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration...

  1. Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    Callesen, Anne K; Christensen, René Depont; Madsen, Jonna S;

    2008-01-01

    Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum...

  2. Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7

    Hennig Lars

    2010-10-01

    Full Text Available Abstract Background The clock-controlled RNA-binding protein AtGRP7 influences circadian oscillations of its own transcript at the post-transcriptional level. To identify additional targets that are regulated by AtGRP7, transcript profiles of transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox and wild type plants were compared. Results Approximately 1.4% of the transcripts represented on the Affymetrix ATH1 microarray showed changes in steady-state abundance upon AtGRP7 overexpression. One third of the differentially expressed genes are controlled by the circadian clock, and they show a distinct bias of their phase: The up-regulated genes preferentially peak around dawn, roughly opposite to the AtGRP7 peak abundance whereas the down-regulated genes preferentially peak at the end of the day. Further, transcripts responsive to abiotic and biotic stimuli were enriched among AtGRP7 targets. Transcripts encoding the pathogenesis-related PR1 and PR2 proteins were elevated in AtGRP7-ox plants but not in plants overexpressing AtGRP7 with a point mutation in the RNA-binding domain, indicating that the regulation involves RNA binding activity of AtGRP7. Gene set enrichment analysis uncovered components involved in ribosome function and RNA metabolism among groups of genes upregulated in AtGRP7-ox plants, consistent with its role in post-transcriptional regulation. Conclusion Apart from regulating a suite of circadian transcripts in a time-of-day dependent manner AtGRP7, both directly and indirectly, affects other transcripts including transcripts responsive to abiotic and biotic stimuli. This suggests a regulatory role of AtGRP7 in the output of the endogenous clock and a complex network of transcripts responsive to external stimuli downstream of the AtGRP7 autoregulatory circuit.

  3. (E)-Propyl α-Cyano-4-Hydroxyl Cinnamylate: A High Sensitive and Salt Tolerant Matrix for Intact Protein Profiling by MALDI Mass Spectrometry

    Wang, Sheng; Xiao, Zhaohui; Xiao, Chunsheng; Wang, Huixin; Wang, Bing; Li, Ying; Chen, Xuesi; Guo, Xinhua

    2016-04-01

    Low-abundance samples and salt interference are always of great challenges for the practical protein profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Herein, a series of carboxyl-esterified derivatives of α-cyano-4-hydroxycinnamic acid (CHCA) were synthesized and evaluated as matrices for MALDI-MS analysis of protein. Among them, (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) was found to exhibit excellent assay performance for intact proteins by improving the detection sensitivity 10 folds compared with the traditional matrices [i.e., super2,5-dihydroxybenzoic acid (superDHB), sinapic acid (SA), and CHCA]. In addition, CHCA-C3 was shown to have high tolerance to salts, the ion signal of myoglobin was readily detected even in the presence of urea (8 M), NH4HCO3 (2 M), and KH2PO4 (500 mM), meanwhile sample washability was robust. These achievements were mainly attributed to improved ablation ability and increased hydrophobicity or affinity of CHCA-C3 to proteins in comparison with hydrophilic matrixes, leading to more efficient ionization of analyte. Furthermore, direct analysis of proteins from crude egg white demonstrated that CHCA-C3 was a highly efficient matrix for the analysis of low-abundance proteins in complex biological samples. These outstanding performances indicate the tremendous potential use of CHCA-C3 in protein profiling by MALDI-MS.

  4. Activity Based Costing versus Traditional Technique

    Dragomirescu Simona Elena; Solomon Daniela Cristina

    2011-01-01

    One of the current methods of management is Activity-Based Costing (ABC), method that allows the company to understand more clearly how and on what activity/product profit is achieved. In essence, the method involves identifying all specific activities of a product or service and distribution expenses to achieve them with greater accuracy than with traditional accounting methods. This involves not only costs determining closer to reality, but a better understanding of the factors that determi...

  5. Ewolucja koncepcji Activity-Based Costing

    Szychta, Anna

    1997-01-01

    Over the past few years Activity-Based Costing has come to be one of the most popular approaches to management accounting in the United States, Great Britain and many other western countries. According to the ABC conception, indirect costs are allocated to products relative to activities and processes incurring these costs, instead of the classification by manufacturing sub-units, eg. departments, using different bases of costs repatriation, mostly not proportional to the pr...

  6. Expression profiles of 12 late embryogenesis abundant protein genes from Tamarix hispida in response to abiotic stress.

    Gao, Caiqiu; Liu, Yali; Wang, Chao; Zhang, Kaimin; Wang, Yucheng

    2014-01-01

    Twelve embryogenesis abundant protein (LEA) genes (named ThLEA-1 to -12) were cloned from Tamarix hispida. The expression profiles of these genes in response to NaCl, PEG, and abscisic acid (ABA) in roots, stems, and leaves of T. hispida were assessed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). These ThLEAs all showed tissue-specific expression patterns in roots, stems, and leaves under normal growth conditions. However, they shared a high similar expression patterns in the roots, stems, and leaves when exposed to NaCl and PEG stress. Furthermore, ThLEA-1, -2, -3, -4, and -11 were induced by NaCl and PEG, but ThLEA-5, -6, -8, -10, and -12 were downregulated by salt and drought stresses. Under ABA treatment, some ThLEA genes, such as ThLEA-1, -2, and -3, were only slightly differentially expressed in roots, stems, and leaves, indicating that they may be involved in the ABA-independent signaling pathway. These findings provide a basis for the elucidation of the function of LEA genes in future work. PMID:25133264

  7. Identification and expression profiles of neuropeptides and their G protein-coupled receptors in the rice stem borer Chilo suppressalis.

    Xu, Gang; Gu, Gui-Xiang; Teng, Zi-Wen; Wu, Shun-Fan; Huang, Jia; Song, Qi-Sheng; Ye, Gong-Yin; Fang, Qi

    2016-01-01

    In insects, neuropeptides play important roles in the regulation of multiple physiological processes by binding to their corresponding receptors, which are primarily G protein-coupled receptors (GPCRs). The genes encoding neuropeptides and their associated GPCRs in the rice stem borer Chilo suppressalis were identified by a transcriptomic analysis and were used to identify potential targets for the disruption of physiological processes and the protection of crops. Forty-three candidate genes were found to encode the neuropeptide precursors for all known insect neuropeptides except for arginine-vasopressin-like peptide (AVLP), CNMamide, neuropeptide-like precursors 2-4 (NPLP2-4), and proctolin. In addition, novel alternative splicing variants of three neuropeptide genes (allatostatin CC, CCHamide 1, and short neuropeptide F) are reported for the first time, and 51 putative neuropeptide GPCRs were identified. Phylogenetic analyses demonstrated that 44 of these GPCRs belong to the A-family (or rhodopsin-like), 5 belong to the B-family (or secretin-like), and 2 are leucine-rich repeat-containing GPCRs. These GPCRs and their likely ligands were also described. qRT-PCR analyses revealed the expression profiles of the neuropeptide precursors and GPCR genes in various tissues of C. suppressalis. Our study provides fundamental information that may further our understanding of neuropeptidergic signaling systems in Lepidoptera and aid in the design of peptidomimetics, pseudopeptides or small molecules capable of disrupting the physiological processes regulated by these signaling molecules and their receptors. PMID:27353701

  8. Quality properties and expression profiling of protein disulfide isomerase genes during grain development of three spring wheat near isogenic lines

    Dong Liwei

    2016-01-01

    Full Text Available Three wheat glutenin near isogenic lines (NILs CB037A, CB037B and CB037C were used to investigate their quality properties and the transcriptional expression profiles of PDI gene family during grain development. Our purpose is to understand the relationships between the dynamic expression of different PDI genes and glutenin allelic compositions related to gluten quality. The results showed that glutenin allelic variations had no significant effects on main agronomic traits and yield performance, but resulted in clear gluten quality changes. CB037B with 5+10 subunits had higher glutenin macropolymer (GMP content and better breadmaking quality than CB037A with 2+12 while the lack of Glu-B3h encoding one abundant B-subunit in CB037C significantly reduced GMP content, dough strength and breadmaking quality. The dynamic expression patterns of eight protein disulfide isomerase (PDI genes during grain development detected by quantitative real-time polymerase chain reaction (qRT-PCR showed the close correlations between higher expression levels of PDI3-1, PDI5-1 and PDI8-1 and the presence of 5+10 subunits. Meanwhile, Glu-B3h silence resulted in significant decrease of expression levels of five PDI genes (PDI3-1, PDI5-1, PDI6-1, PDI7-2 and PDI8-1, suggesting the vital roles of certain PDI genes in glutenin and GMP synthesis and gluten quality formation.

  9. Comparative Electrophoretic Study of Protein Profiles of Normal and Sterile Medfly, Ceratitis capitata Wied. Treated with Various Concentrations of E-Selen Antioxidant

    The dietary effects of E-Selen on the protein profiles of normal and irradiated medfly (Ceratitis capitata Wied .), were investigated in total body homogenates of the pupae. The insects were reared from first-instar larvae on artificial diets containing 0, 0.1, 0.3, 0.5 and 1.5 mg of E-Selen. The appearance and disappearance of some high molecular weight protein fractions among the different samples tested due to the difference of antioxidant doses were detected. All bands of molecular weights ranging between 51.5 and 9.9 KDa were commonly found among all samples while the variations were detected only in the bands of higher molecular weights. Disappearance of some protein bands indicated may be due to non-production, utilization or the degradation of proteins to maintain amino acids concentrations in the body, while the appearance of new bands might indicate the synthesis of new proteins by the insect

  10. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity

    Zulezwan A. Malik

    2013-12-01

    Full Text Available Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC coupled to mass spectrometry (MS affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group bred as either high- or low-capacity runners (HCR and LCR, respectively that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001 in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897 and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5. Sixteen proteins were significantly (p < 0.05 more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH was 1

  11. Balanced activity scorecard – combination of activity based costing and activity based management with balanced scorecard

    Dorota Kuchta; Radoslaw Rynca

    2006-01-01

    The paper presents a proposal of the Activity Balance Scorecard (ABSC). It is a combination of Activity Based Costing and a modification of Activity Based Management. Contrary to the traditional cascading of the Balanced Scorecard to organisational structures, ABSC is constructed directly for activities or tasks. These activities or tasks should be selected on the basis of ABC results – as it is them which give the information the share of which tasks in the cost structure is high. The ABSC w...

  12. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products.

    Hegele, Jörg; Buetler, Timo; Delatour, Thierry

    2008-06-01

    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products. PMID:18486644

  13. Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

    Xiaolin Wu

    Full Text Available Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs, mainly mannose-binding lectin (agglutinin, exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE. Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

  14. Association of -971 G/A Cholesteryl-Ester Transfer Protein Gene Polymorphism with Lipid Profile in Primary Hyperlipidemia

    A Barkhordari

    2012-10-01

    Full Text Available Background: Coronary heart disease (CHD is a leading cause of death worldwide and hypertriglyceridemia and hypercholesterolemia are major risk factors for the disease. Considering the role of hyperlipidemia as the underlying cause of cardiovascular mortalities and morbidities, and the limited and conflicting results of studies on CETP gene polymorphisms in Iran, we aimed to study -971 G/A polymorphism of cholesterol ester transfer protein gene in patients with primary hyperlipidemia.Methods: In this case-control study performed in Hamadan University of Medical Sciences (from May 2010 to April 2011, we recruited 200 patients with primary hyperlipidemia (total cholesterol >250 mg/dl and/or triglyceride >200 mg/dl as the cases and 200 healthy individuals with normal cholesterol and triglyceride as the control group. Gene segments were replicated by polymerase chain reaction (PCR and -971 G/A polymorphism genotypes were identified by RFLP technique. Subsequently, plasma CETP activity was measured enzymeatically by a kit in a fluorescence spectrometer.Results: The allele and genotype frequencies were not significantly different (P>0.05 between the two groups (in the control group: AA 24%, GA 47% and GG 28.5% and in the case group: AA 18%, GA 51% and GG 31%. In the case group, homozygous individuals with A alleles (AA genotype had greater cholesterol and HDL-c concentrations than those with other alleles (GG and GA. In both cases and controls, individuals with AA genotype had lower CETP concentrations.Conclusion: We conclude that -971 G/A polymorphism in CETP gene promoter can affect lipid profile and alter CETP activity.

  15. Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori.

    Dong, Zhaoming; Wang, Xiaohuan; Zhang, Yan; Zhang, Liping; Chen, Quanmei; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

    2016-05-01

    Male accessory gland (MAG) and female accessory gland (FAG) of the reproductive system are, respectively, responsible for producing seminal proteins and adhesive proteins during copulation and ovulation. Seminal proteins are ejaculated to female along with sperms, whereas adhesive proteins are excreted along with eggs. Proteins from the male and female reproductive organs are usually indicative of rapid adaptive evolution. Understanding the reproductive isolation and species divergence requires identifying reproduction-related proteins from many different species. Here, we present our proteomic analyses of male and female accessory glands of the silkworm, Bombyx mori. Using LC/MS-MS, we identified 2133 MAG proteins and 1872 FAG proteins. In total, 652 proteins were significant more abundant in the MAG than in the FAG, including growth factors, odorant-binding proteins, enzymes, and proteins of unknown function. Growth factors and odorant-binding proteins are potential signaling molecules, whereas most of proteins of unknown function were found to be Lepidoptera-specific proteins with high evolutionary rates. Microarray experiments and semi-quantitative RT-PCR validated that MAG-specific proteins were expressed exclusively in male moths. Totally, 192 proteins were considered as FAG-specific proteins, including protease inhibitors, enzymes, and other proteins. Protease inhibitors were found to be the most abundant FAG-specific proteins, which may protect eggs from infection by inhibiting pathogen-derived proteases. These results provide comprehensive insights into copulation and oviposition. Moreover, the newly identified Lepidoptera-specific MAG proteins provide useful data for future research on the evolution of reproductive proteins in insects. PMID:26822097

  16. Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis

    Eroukova Veronika

    2008-12-01

    Full Text Available Abstract Background Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains for increased sensitivity to paromomycin, which targets the process of protein synthesis. Results As expected, our analysis indicated that the majority of deletion strains (134 with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45, cellular component biogenesis and organization (28, DNA maintenance (21, transport (20, others (38 and unknown (39. These may represent minor cellular target sites (side-effects for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. Conclusion We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s.

  17. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  18. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p ion (551.21 m/z) of the doubly-charged peptide SLGVGFATR (454.19 m/z) of residues 23-31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater (p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater (p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for earlier

  19. Low gamma irradiation effects on protein profile, solubility, oxidation, scavenger ability and bioavailability of essential minerals in black and yellow Indian soybean (Glycine max L.) varieties

    Effect of low doses of gamma irradiation (0.25, 0.5 and 1.0 kGy) on protein oxidation, profile, solubility, ROS scavenging and in vivo bioavailability of minerals in black (BS1) and yellow (BRAGG) soybean varieties were investigated. Increased oxidation, altered protein profile with decreased solubility was observed higher in BRAGG compared with BS1. The most significant ROS scavenging effect, antioxidant activity, least phytate content and improved bioavailability was found at 0.5 kGy in BS1 than BRAGG due to anthocyanins, and phenolics. Still 1.0 kGy is considered as toxicologically and microbiologically safe but it causes biochemical alterations and thus 0.5 kGy can be the optimum dose with enriched nutraceutical properties. (author)

  20. Searching for early breast cancer biomarkers by serum protein profiling of pre-diagnostic serum; a nested case-control study

    Serum protein profiles have been investigated frequently to discover early biomarkers for breast cancer. So far, these studies used biological samples collected at or after diagnosis. This may limit these studies' value in the search for cancer biomarkers because of the often advanced tumor stage, and consequently risk of reverse causality. We present for the first time pre-diagnostic serum protein profiles in relation to breast cancer, using the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort. In a nested case-control design we compared 68 women diagnosed with breast cancer within three years after enrollment, with 68 matched controls for differences in serum protein profiles. All samples were analyzed with SELDI-TOF MS (surface enhanced laser desorption/ionization time-of-flight mass spectrometry). In a subset of 20 case-control pairs, the serum proteome was identified and relatively quantified using isobaric Tags for Relative and Absolute Quantification (iTRAQ) and online two-dimensional nano-liquid chromatography coupled with tandem MS (2D-nanoLC-MS/MS). Two SELDI-TOF MS peaks with m/z 3323 and 8939, which probably represent doubly charged apolipoprotein C-I and C3a des-arginine anaphylatoxin (C3adesArg), were higher in pre-diagnostic breast cancer serum (p = 0.02 and p = 0.06, respectively). With 2D-nanoLC-MS/MS, afamin, apolipoprotein E and isoform 1 of inter-alpha trypsin inhibitor heavy chain H4 (ITIH4) were found to be higher in pre-diagnostic breast cancer (p < 0.05), while alpha-2-macroglobulin and ceruloplasmin were lower (p < 0.05). C3adesArg and ITIH4 have previously been related to the presence of symptomatic and/or mammographically detectable breast cancer. We show that serum protein profiles are already altered up to three years before breast cancer detection

  1. Nosocomial klebsiella infection in neonates in a tertiary care hospital: Protein profile by SDS-page and klebocin typing as epidemiological markers

    Malik A; Hasani S; Shahid M; Khan H; Ahmad A

    2003-01-01

    PURPOSE: To find out the prevalence of Klebsiella in hospital acquired neonatal infections in a tertiary care set up and to evaluate the role of klebocin typing and protein profile by SDS-PAGE in epidemiological typing of the isolates. METHODS: Hospital born neonates transferred to the neonatal unit after birth and available in the unit 48 hours later comprised the study group. Two hundred and three neonates were found eligible for inclusion in the study. Repeated blood cultures, other releva...

  2. Modulating protein release profiles by incorporating hyaluronic acid into PLGA microparticles Via a spray dryer equipped with a 3-fluid nozzle

    Wan, Feng; Maltesen, Morten Jonas; Andersen, Sune Klint;

    2014-01-01

    with or without HA were prepared using a spray dryer equipped with a 3-fluid nozzle. The effects of HA on the surface tension and the rheological behavior of the inner feed solution were investigated. The physicochemical properties of the resulting microparticles were characterized using scanning electron......: The present work demonstrates the potential of HA to modulate protein release profile from PLGA microparticle formulations produced via spray drying using 3-fluid nozzle....

  3. Nucleic acid labeling with [3H]orotic acid and nucleotide profile in rats in protein deprivation, enteral and parenteral essential amino acid administration, and 5-fluorouracil treatment

    Rats were fed a 0% casein diet for 1 week, with or without enteral or parenteral administration of essential amino acids, or a 25% casein diet, in one group supplemented with 5-fluorouracil treatment. Ninety minutes before sacrifice the rats were given a tracer of [3H]orotic acid. Incorporation into the acid soluble fraction, RNA, and DNA was determined in liver, small intestine, bone marrow, and kidney. Nucleotide profile was examined in liver and intestine. Protein deficiency caused inter alia a decrease in body weight; a decrease in RNA/DNA ratio and an increase in the specific RNA labeling in liver and kidney; an altered nucleotide profile in the liver; an increase in the nucleotide/DNA and RNA/DNA ratios and a decrease in the specific labeling of the acid soluble fraction, RNA, and DNA in the bone marrow. These changes were prevented to the same extent by giving essential amino acids, either orally or intravenously. The minor changes in intestinal nucleotide profile in protein deprivation were prevented to a slightly larger extent by amino acids orally than parenterally. 5-Fluorouracil treatment gave a decrease in the RNA/DNA ratio in the liver and kidney but an increase in the nucleotide/DNA and RNA/DNA ratios in the bone marrow. Nucleotide profiles were unaltered. The amount of DNA per gram of tissue decreased in bone marrow and increased in kidney. Parenteral administration per se resulted in almost no changes

  4. Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

    Chun-Hong Zhao; Qi-Fu Li

    2005-01-01

    AIM: To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy.METHODS: Nuclear matrix proteins were selectively extracted from MGc80-3 cells treated with or without hexamethylamine bisacetamide (HMBA), and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis and submitted for database searching using Mascot tool.RESULTS: The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGc80-3 cells compared to control.Eleven of which were identified. Seven proteins -actin, prohibitin, porin 31HL, heterogeneous nuclear ribonucleoprotein A2/B1, vimentin, ATP synthase, and heatshock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated,and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells.CONCLUSION: The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

  5. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  6. Activity Based Costings anvendelse til beslutningstagen

    Nielsen, Steen

    2007-01-01

    Activity-Based Costing (ABC) er stadig ét af mest omtalte "moderne" økonomistyringsværktøjer såvel blandt private virksomheder af undersøgelsen, som i offentlige organisationer. En ny state-of-the-art undersøgelse af 90 mellemstore og større danske fremstillingsvirksomheder blev gennemført i 2003...... relation til de fremtidige muligheder, herunder en cost/benefit betragtning ved implementering af ABC. Vi har tilføjet en del kvalitative kommentarer, som respondenterne har tilføjet. Disse er vigtige i forhold til en forståelse for, hvorfor eller hvorfor ikke ABC anvendes. Til sidst perspektiveres også...

  7. Implications of high-temperature events and water deficits on protein profiles in wheat (Triticum aestivum L. cv. Vinjett) grain

    Yang, Fen; Jørgensen, Anders Dysted; Li, Huawei;

    2011-01-01

    proteins involved in primary metabolism, storage and stress response such as late embryogenesis abundant proteins, peroxiredoxins and alpha-amylase/trypsin inhibitors. Several proteins, e.g. heat shock protein and 14-3-3 protein changed in abundance only under multiple high temperatures....... interaction of water deficits and/or a high-temperature event (32 degrees C) during vegetative growth (terminal spikelet) with either of these stress events applied during generative growth (anthesis) in wheat. Influence of combinations of stress on protein fractions (albumins, globulins, gliadins and...... glutenins) in grains and stress-induced changes on the albumin and gliadin proteomes were investigated by 2-DE and MS. The synthesis of individual protein fractions was shown to be affected by both the type and time of the applied stresses. Identified drought or high-temperature-responsive proteins included...

  8. Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don) Endl

    Chen Yu-Ting; Kuo Ching-I; Huang Bau-Lian; Hsu Ya-Wen; Hsieh Tsung-Ju; Shen Chin-Hui; Chen Peng-Jen; Chang Ing-Feng; Chu Hsiu-An; Yeh Kai-Wun; Huang Li-Chun

    2010-01-01

    Abstract Background Restoration of rooting competence is important for rejuvenation in Sequoia sempervirens (D. Don) Endl and is achieved by repeatedly grafting Sequoia shoots after 16 and 30 years of cultivation in vitro. Results Mass spectrometry-based proteomic analysis revealed three proteins that differentially accumulated in different rejuvenation stages, including oxygen-evolving enhancer protein 2 (OEE2), glycine-rich RNA-binding protein (RNP), and a thaumatin-like protein. OEE2 was f...

  9. Quantitative Proteomic Profiling of Muscle Type-Dependent and Age-Dependent Protein Carbonylation in Rat Skeletal Muscle Mitochondria

    Feng, Juan; Xie, Hongwei; Meany, Danni L.; Thompson, LaDora V.; Arriaga, Edgar A.; Griffin, Timothy J.

    2008-01-01

    Carbonylation is a highly prevalent protein modification in skeletal muscle mitochondria, possibly contributing to its functional decline with age. Using quantitative proteomics, we identified mitochondrial proteins susceptible to carbonylation in a muscle type (slow- vs fast-twitch)-dependent and age-dependent manner from Fischer 344 rat skeletal muscle. Fast-twitch muscle contained twice as many carbonylated mitochondrial proteins than did slow-twitch muscle, with 22 proteins showing signif...

  10. New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts

    Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B.; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

    2016-01-01

    Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the

  11. New insight into quinoa seed quality under salinity: changes in proteomic and amino acid profiles, phenolic content, and antioxidant activity of protein extracts

    Iris eAloisi

    2016-05-01

    Full Text Available Quinoa (Chenopodium quinoa Willd is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa seeds. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49 and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR, exposed to two levels of salinity (100 and 300 mM NaCl were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC and flavonoid (TFC contents, and antioxidant activity (AA of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace

  12. New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts.

    Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

    2016-01-01

    Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the

  13. Lipid profile and levels of homocysteine, leptin, fibrinogen and C-reactive protein in hyperthyroid patients before and after treatment

    Emine Sütken

    2010-03-01

    Full Text Available Objectives: The present study was carried out to determine whether thyroid hormones affect lipid profile and levels of erithrocyte sedimentation rate (ESR, serum total homocysteine (t-hcy, leptin, fibrinogen, C-reactive protein (CRP in patients with hyperthyroidism.Materials and methods: This study was carried out on 23 hyperthroid subjects (3 men / 20 women, mean age 41.8 ± 2.4 years. Serum levels of homocysteine, leptin, fibrinogen, CRP, total cholesterol (TC, high-density lipoprotein cholesterol (HDL-C, low-density lipoprotein cholesterol (LDL-C and ESR were measured and body mass index (BMI were calculated before and after treatment of hyperthyroidism.Results: Pretreatment t-hcy, TC, LDL-C, HDL-C levels and BMI of patients were significantly lower than those of the post-treatment (p<0.001, for each variable. However, fibrinogen and ESR decreased after the treatment (p<0.001 and p<0.05, respectively. There were no differences in leptin and CRP levels between pre- and post-treatment periods. Pre and post treatment TC and LDL-C levels were negatively correlated with free triiodothyronine (fT3 levels (r=-0.588, p<0.01; r=-0.534, p<0.01; r=-0.543, p<0.01 and r =-0.653, p<0.01, respectively. Pre-treatment HDL-C was inversely correlated with TSH (r=-0.423, p<0.05. Pre-post- treatment LDL-C was negatively correlated with free thyroxine (fT4 levels (r=-0.536, p<0.001 and r=- 0.422, p<0.05 respectively. Pre-treatment TC was inversely correlated with fT4 (r=-0.590, p<0.01.Conclusion: Hyperthyroidism is associated with high plasma fibrinogen and ESR levels. Elevated plasma fibrinogen and ESR levels may be a possible explanation for the high cardiovascular morbidity among hyperthyroidic subjects. These changes may reflect low-grade inflammation or disturbances in coagulation in hyperthyroidism.

  14. Expression profile of G-protein βγ subunit gene transcripts in the mouse olfactory sensory epithelia

    Aaron eSathyanesan

    2013-06-01

    Full Text Available Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE and the vomeronasal organ (VNO. Our reverse transcriptase PCR (RT-PCR and realtime qPCR analyses of all known Gβ(β1,2,3,4,5 and Gγ(γ1,2,2t,3,4,5,7,8,10,11,12,13 subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs, while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβsubunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3 and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7 and 14 and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system.

  15. Correlation between Phylogroups and Intracellular Proteomes of Propionibacterium acnes and Differences in the Protein Expression Profiles between Anaerobically and Aerobically Grown Cells

    Itaru Dekio

    2013-01-01

    Full Text Available Propionibacterium acnes is one of the dominant commensals on the human skin and also an opportunistic pathogen in relation to acne, sarcoidosis, prostate cancer, and various infections. Recent investigations using housekeeping and virulence genes have revealed that the species consists of three major evolutionary clades (types I, II, and III. In order to investigate protein expression differences between these phylogroups, proteomic profiles of 21 strains of P. acnes were investigated. The proteins extracted from cells cultured under anaerobic and aerobic conditions were analysed using a SELDI-TOF mass spectrometer, high-resolution capillary gel electrophoresis, and LC-MS/ MS. The SELDI spectral profiles were visualised as a heat map and a dendrogram, which resulted in four proteomic groups. Strains belonging to type I were represented in the proteome Group A, while Group B contained type III strains. Groups C and D contained mixtures of types I and II. Each of these groups was not influenced by differences in culture conditions. Under anoxic growth conditions, a type IB strain yielded high expressions of some proteins, such as methylmalonyl-CoA epimerase and the Christie-Atkins-Munch-Petersen (CAMP factor. The present study revealed good congruence between genomic and proteomic data suggesting that the microenvironment of each subtype may influence protein expression.

  16. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck;

    2012-01-01

    It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is...... labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited...... more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel...

  17. Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2016-09-01

    Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. PMID:27150797

  18. Einsatz von Protein- und Metabolit-Profiling-Methoden zur Unterscheidung von ökologischem und konventionellem Weizen

    Bonte, Anja; Kessler, Nikolas; Nattkemper, Tim; Goesmann, Alexander; Thonar, Cécile; Mäder, Paul; Niehaus, Karsten; Langenkämper, Georg

    2013-01-01

    The interest in methods to proof organic food authenticity increases with the steadily rising popularity of food labelled organic. Profiling techniques enable the detection of a wide range of substances in biological samples. Together with bioinformatics tools these techniques are useful for biomarker searching, e. g. in plant extracts. Metabolomic and proteomic profiling techniques were used to screen organic and conventional wheat, originating from the DOK field trial in Switzerland. Up to ...

  19. Salt-induced root protein profile changes in seedlings of maize inbred lines with differing salt tolerances

    Yujing Cheng

    2014-12-01

    Full Text Available Salt stress is one of the severest growth limited-factors to agriculture production. To gain in-depth knowledge of salt-stress response mechanisms, the proteomics analysis from two maize (Zea mays L. inbred lines was carried out using two-dimensional gel electrophoresis (2-DGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS. There were 57 salt-regulated proteins identified, 21 and 36 proteins were differentially regulated in inbred lines 'Nongda 1145' (salt-resistant and 'D340' (salt-sensitive, respectively. The identified proteins were distributed in 11 biological processes and seven molecular functions. Under salt stress, proteins related to antioxidation and lignin synthesis were increased in both inbred lines. The relative abundance of proteins involved in translation initiation, elongation, and protein proteolysis increased in 'Nongda 1145' and decreased in 'D340'. In addition, the abundance of proteins involved in carbohydrate metabolism, protein refolding, ATP synthase and transcription differed between the two inbred lines. Our results suggest that the enhanced ability of salt-tolerant inbred line 'Nongda 1145' to combat salt stress occurs via regulation of transcription factors promoting increased antioxidation and lignin biosynthesis, enhanced energy production, and acceleration of protein translation and protein proteolysis.

  20. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

    Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

    2016-09-01

    This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d. PMID:27490632

  1. Amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key foods in Bangladesh.

    Shaheen, Nazma; Islam, Saiful; Munmun, Sarah; Mohiduzzaman, Md; Longvah, Thingnganing

    2016-12-15

    Concentrations of standard amino acids were determined in the composite samples (representing 30 agro-ecological zones of Bangladesh) of six prioritized key dietary protein sources: Oryza sativa (rice), Triticum aestivum (wheat flour), Lens culinaris (lentils), Pangusius pangusius (pangas), Labeo rohita (rohu) and Oreochromis mossambicus (tilapia). Digestible indispensable amino acid scores (DIAAS) was calculated using published data on amino acids' digestibility to evaluate the protein quality of these foods. Indispensable amino acid (IAA) contents (mg IAA/g protein), found to be highest in pangas (430) and lowest in wheat (336), of all these analyzed foods exceeded the FAO recommended daily allowance (277mg IAA/g protein) and contributed on average 40% to total amino acid contents. Untruncated DIAAS values ranged from 51% (lysine) in wheat to 106% (histidine) in pangas and distinguished pangas, rohu, and tilapia containing 'excellent quality' protein (DIAAS>100%) with potential to complement lower quality protein of cereals, fruits, and vegetables. PMID:27451158

  2. A chromatin activity based chemoproteomic approach reveals a transcriptional repressome for gene-specific silencing

    Liu, Cui; Yu, Yanbao; Liu, Feng; Wei, Xin; Wrobel, John A; Gunawardena, Harsha P.; Zhou, Li; Jin, Jian; Chen, Xian

    2014-01-01

    Immune cells develop endotoxin tolerance (ET) after prolonged stimulation. ET increases the level of a repression mark H3K9me2 in the transcriptional-silent chromatin specifically associated with pro-inflammatory genes. However, it is not clear what proteins are functionally involved in this process. Here we show that a novel chromatin activity based chemoproteomic (ChaC) approach can dissect the functional chromatin protein complexes that regulate ET-associated inflammation. Using UNC0638 th...

  3. Mesothelioma: profile of keratin proteins and carcinoembryonic antigen: an immunoperoxidase study of 20 cases and comparison with pulmonary adenocarcinomas.

    Corson, J M; Pinkus, G. S.

    1982-01-01

    The distribution of keratin proteins and carcinoembryonic antigen (CEA) in 20 diffuse pleural malignant mesotheliomas and 20 adenocarcinomas of the lung was determined with the use of an indirect immunoperoxidase method. Keratin proteins were identified in all of the mesotheliomas, with strong staining observed in 17 of the cases. Tumor cells of various histologic types (tubular, papillary, solid, and spindle) revealed staining for keratin proteins. A variety of staining patterns were observe...

  4. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for Nε-fructoselysine (FL), Nε-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lysfree) and 81.5 ± 87.8 nmol Pyr μmol-1 Lysfree-1 vs. 3.72 ± 1.29 nmol FL μmol-1 Lysfree-1. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol-1 of protein-bound Lys (Lysp-b), 0.04 ± 0.03 nmol CML μmol-1 Lysp-b-1 and 0.06 ± 0.02 nmol Pyr μmol-1 Lysp-b-1. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products

  5. FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles.

    Schneidman-Duhovny, Dina; Hammel, Michal; Tainer, John A; Sali, Andrej

    2016-07-01

    Small Angle X-ray Scattering (SAXS) is an increasingly common and useful technique for structural characterization of molecules in solution. A SAXS experiment determines the scattering intensity of a molecule as a function of spatial frequency, termed SAXS profile. Here, we describe three web servers for modeling atomic structures based on SAXS profiles. FoXS (Fast X-Ray Scattering) rapidly computes a SAXS profile of a given atomistic model and fits it to an experimental profile. FoXSDock docks two rigid protein structures based on a SAXS profile of their complex. MultiFoXS computes a population-weighted ensemble starting from a single input structure by fitting to a SAXS profile of the protein in solution. We describe the interfaces and capabilities of the servers (salilab.org/foxs), followed by demonstrating their application on Interleukin-33 (IL-33) and its primary receptor ST2. PMID:27151198

  6. Identification and Expression Profiling of the BTB Domain-Containing Protein Gene Family in the Silkworm, Bombyx mori

    Daojun Cheng

    2014-01-01

    Full Text Available The BTB domain is a conserved protein-protein interaction motif. In this study, we identified 56 BTB domain-containing protein genes in the silkworm, in addition to 46 in the honey bee, 55 in the red flour beetle, and 53 in the monarch butterfly. Silkworm BTB protein genes were classified into nine subfamilies according to their domain architecture, and most of them could be mapped on the different chromosomes. Phylogenetic analysis suggests that silkworm BTB protein genes may have undergone a duplication event in three subfamilies: BTB-BACK-Kelch, BTB-BACK-PHR, and BTB-FLYWCH. Comparative analysis demonstrated that the orthologs of each of 13 BTB protein genes present a rigorous orthologous relationship in the silkworm and other surveyed insects, indicating conserved functions of these genes during insect evolution. Furthermore, several silkworm BTB protein genes exhibited sex-specific expression in larval tissues or at different stages during metamorphosis. These findings not only contribute to a better understanding of the evolution of insect BTB protein gene families but also provide a basis for further investigation of the functions of BTB protein genes in the silkworm.

  7. GprotPRED: Annotation of Gα, Gβ and Gγ subunits of G-proteins using profile Hidden Markov Models (pHMMs) and application to proteomes.

    Kostiou, Vasiliki D; Theodoropoulou, Margarita C; Hamodrakas, Stavros J

    2016-05-01

    Heterotrimeric G-proteins form a major protein family, which participates in signal transduction. They are composed of three subunits, Gα, Gβ and Gγ. The Gα subunit is further divided in four distinct families Gs, Gi/o, Gq/11 and G12/13. The goal of this work was to detect and classify members of the four distinct families, plus the Gβ and the Gγ subunits of G-proteins from sequence alone. To achieve this purpose, six specific profile Hidden Markov Models (pHMMs) were built and checked for their credibility. These models were then applied to ten (10) proteomes and were able to identify all known G-protein and classify them into the distinct families. In a separate case study, the models were applied to twenty seven (27) arthropod proteomes and were able to give more credible classification in proteins with uncertain annotation and in some cases to detect novel proteins. An online tool, GprotPRED, was developed that uses these six pHMMs. The sensitivity and specificity for all pHMMs were equal to 100% with the exception of the Gβ case, where sensitivity equals to 100%, while specificity is 99.993%. In contrast to Pfam's pHMM which detects Gα subunits in general, our method not only detects Gα subunits but also classifies them into the appropriate Gα-protein family and thus could become a useful tool for the annotation of G-proteins in newly discovered proteomes. GprotPRED online tool is publicly available for non-commercial use at http://bioinformatics.biol.uoa.gr/GprotPRED and, also, a standalone version of the tool at https://github.com/vkostiou/GprotPRED. PMID:26854601

  8. Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples

    Mark J Bailey

    2013-01-01

    Full Text Available Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4. The expression of TPM4 increased by 2-fold in Stage 2 before returning to "normal" levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage.

  9. Evaluation of RAPD-PCR and protein profile analysis to differentiate Vibrio harveyi strains prevalent along the southwest coast of India

    Biswajit Maiti; Malathi Shekar; Rekha Khushiramani; Iddya Karunasagar; Indrani Karunasagar

    2009-12-01

    Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index $(D)$ value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.

  10. Protein profile of basal prostate epithelial progenitor cells-stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

    Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

    2016-04-01

    The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-) Sca-1(+) CD49f(+) Trop2(high) -phenotype) and human (Lin(-) CD49f(+) TROP2(high) ) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+) /TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+) /CD24(+) /CD29(+) /CD44(+) /CD47(+) /CD49f(+) /CD104(+) /CD147(+) /CD326(+) /Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. PMID:26849468

  11. A peptidomic approach to study the contribution of added casein proteins to the peptide profile in Spanish dry-fermented sausages.

    Mora, Leticia; Escudero, Elizabeth; Aristoy, M-Concepción; Toldrá, Fidel

    2015-11-01

    Peptidomics is a necessary alternative in the analysis of naturally generated peptides in dry-fermented processing. The intense proteolysis occurred during the processing of dry-fermented sausages is due to the action of endopeptidases and exopeptidases from both, endogenous muscle origin and lactic acid bacteria (LAB) added in the starter. Sodium caseinate is frequently used as an additive in this type of products because of its emulsifying properties, and consequently influences the protein profile available during the proteolysis. In this study, a mass spectrometry approach has been used to determine the impact of added sodium caseinate in the final peptide profile as well as to analyse its possible influence in the presence of certain previously described casein-derived bioactive peptides. PMID:26116420

  12. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

    Pedersen, K.; Gram, Lone; Austin, D.A.;

    1997-01-01

    The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only str...

  13. Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael;

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied t...

  14. Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance spectroscopy

    Cruz, A; Casals, C; Plasencia, I;

    1998-01-01

    Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for t...

  15. Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

    Suh Moo-Jin

    2012-04-01

    Full Text Available Abstract Background The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. Results To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210, was also one of the smallest proteins detected in this study (M.W. 7,367. Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for

  16. Effect of the addition of flavan-3-ols on the HPLC-DAD salivary-protein profile.

    Quijada-Morín, Natalia; Crespo-Expósito, Carlos; Rivas-Gonzalo, Julián C; García-Estévez, Ignacio; Escribano-Bailón, María Teresa

    2016-09-15

    The interaction between monomeric flavan-3-ols and salivary proteins has been studied using HPLC-DAD. A chromatographic method has been described and seven protein fractions were collected. The peptides and proteins present in each fraction have been identified using nLC-MS-MS analysis. The interaction between saliva and catechin, epicatechin and gallocatechin has been studied. These compounds interact in a discriminated way with salivary proteins: catechin causes a decrease of some fractions, epicatechin causes the decrease or increase of fractions while gallocatechin seems to cause an increase of two fractions. This variable behavior is explained, for the decrease in the chromatographic area, by the precipitation of salivary proteins and, for the increase of the area, by the formation of soluble complexes and/or for the formation of new peaks. PMID:27080905

  17. Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas

    Begcevic, Ilijana; Brinc, Davor; Drabovich, Andrei P.; Batruch, Ihor; Diamandis, Eleftherios P.

    2016-01-01

    Background Cerebrospinal fluid (CSF) is a proximal fluid which communicates closely with brain tissue, contains numerous brain-derived proteins and thus represents a promising fluid for discovery of biomarkers of central nervous system (CNS) diseases. The main purpose of this study was to generate an extensive CSF proteome and define brain-related proteins identified in CSF, suitable for development of diagnostic assays. Methods Six non-pathological CSF samples from three female and three mal...

  18. LEVELS OF BRAIN-SPECIFIC S-100B PROTEIN, SPECIFIC ANTIBODIES AND CYTOKINE PROFILE IN THE PATIENTS WITH ALCOHOL-INDUCED DELIRIUM STATES

    N. N. Tsybikov

    2008-01-01

    Full Text Available Abstract. Present article deals with our results concerning brain-specific S-100B protein levels, anti-S-100B autoantibodies of IgM and IgG classes, like as cytokine profiles of blood serum and cerebrospinal fluid in the patients with alcohol-induced delirium state. The results obtained provide an evidence of association between alcoholic psychosis and destruction of brain tissue, development of autoimmune reactions and altered cytokine status, thus, probably, resulting into disintegration of immune and neuroendocrine systems.

  19. Redox proteomic profiling of neuroketal-adducted proteins in human brain: Regional vulnerability at middle age increases in the elderly.

    Domínguez, Mayelín; de Oliveira, Eliandre; Odena, María Antonia; Portero, Manuel; Pamplona, Reinald; Ferrer, Isidro

    2016-06-01

    Protein lipoxidation was assessed in the parietal cortex (PC), frontal cortex (FC), and cingulate gyrus (CG) in middle-aged and old-aged individuals with no clinical manifestations of cognitive impairment, in order to increase understanding of regional brain vulnerability to oxidative damage during aging. Twenty-five lipoxidized proteins were identified in all the three regions although with regional specificities, by using redox proteomics to detect target proteins of neuroketals (NKT) adduction. The number of cases with NKT-adducted proteins was higher in old-aged individuals but most oxidized proteins were already present in middle-aged individuals. Differences in vulnerability to oxidation were dependent on the sub-cellular localization, secondary structure, and external exposition of certain amino acids. Lipoxidized proteins included those involved in energy metabolism, cytoskeleton, proteostasis, neurotransmission and O2/CO2, and heme metabolism. Total NKT and soluble oligomer levels were estimated employing slot-blot, and these were compared between age groups. Oligomers increased with age in PC and FC; NKT significantly increased with age in FC, whereas total NKT and oligomer levels were not modified in CG, thus highlighting differences in brain regional vulnerability with age. Oligomers significantly correlated with NKT levels in the three cortical regions, suggesting that protein NKT adduction parallels soluble oligomer formation. PMID:26968793

  20. Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

    Kawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, Tohru

    2016-06-17

    N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics. PMID:27010125

  1. Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles.

    Deeb, Sally J; Tyanova, Stefka; Hummel, Michael; Schmidt-Supprian, Marc; Cox, Juergen; Mann, Matthias

    2015-11-01

    Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology. PMID:26311899

  2. Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    Callesen, Anne K; Mohammed, Shabaz; Bunkenborg, Jakob;

    2005-01-01

    Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. Sample preparation is key to achieving reproducible and well-resolved signals in MALDI-MS; a prerequisite for...

  3. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis

    Vogel, Birte Fonnesbech; Jørgensen, K.; Christensen, H.;

    1997-01-01

    Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing...... methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole- cell protein profiling and ribotyping......, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently...

  4. Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

    Gutjahr, C.; Murphy, D.; Lueking, A.; Koenig, A.; Janitz, M; O'Brien, J.; Korn, B. (Bernhard); S. Horn; Lehrach, H; Cahill, D.

    2005-01-01

    The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (TH1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particula...

  5. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-11-30

    Background: High doses of ionizing radiation result in biological damage, however the precise relationships between long term health effects, including cancer, and low dose exposures remain poorly understood and are currently extrapolated using high dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose dependent responses to radiation. Principle Findings: We have identified 6845 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts one hour post-exposure. Dual statistical analyses based on spectral counts and peak intensities identified 287 phosphopeptides (from 231 proteins) and 244 phosphopeptides (from 182 proteins) that varied significantly following exposure to 2 and 50 cGy respectively. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatics analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role of MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conlcusions: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provides a basis for the systems level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at

  6. Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

    Currie, D. H.; Guss, A. M.; Herring, C. D.; Giannone, R. J.; Johnson, C. M.; Lankford, P. K.; Brown, S. D.; Hettich, R.L.; Lynd, L. R.

    2014-01-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases an...

  7. Protein profiling of paraquat-exposed rat lungs following treatment with Acai (Euterpe oleracea Mart.) berry extract.

    Kim, Yong-Sik; Jung, Hana; Zerin, Tamanna; Song, Ho-Yeon

    2013-03-01

    Paraquat (1,1'-dimethyl-4,4'-bipyridinium chloride, PQ) is a non-selective herbicide, and PQ poisoning by accidental or intentional ingestion is a cause of numerous fatalities around the world every year. Although a great deal of research has been conducted into the development of an acceptable treatment for PQ poisoning, no effective guidelines for patients have been developed thus far. Acai berry extract and juice have been highlighted in this regard, due to their observed antioxidant effects in various diseases. Furthermore, the acai berry has been used in dietary supplements, as it contains a variety of nutrients, including proteins, lipids, vitamins A, C and E and polyphenols. In this study, we conducted proteomic analysis of PQ-poisoned rat lungs to evaluate the changes in protein expression induced by PQ and to identify any protective effects of acai berry on the PQ poisoning. Our data revealed that the expression of the calcium signaling-related proteins calcium binding protein 1 (CaBP1), FK506 binding protein 4 (FKBP4), S100A6 and secreted protein acidic and rich in cysteine (Sparc, also known as osteonectin) were induced by PQ treatment and downregulated by acai berry treatment. However, the levels of protein kinase C substrate 80K-H were shown to be downregulated as the result of PQ treatment. Our results indicated that these proteins may function as biomarkers for acute poisoning by PQ exposure. Further studies may be necessary to understand their clinical relevance with regard to PQ poisoning. PMID:23291665

  8. Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins

    Caroline Lewis

    2010-01-01

    Full Text Available Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

  9. Proteomic profiling of glucocorticoid-exposed myogenic cells: Time series assessment of protein translocation and transcription of inactive mRNAs

    Hoffman Eric P

    2009-07-01

    approaches of subcellular proteomic profiling and assessment of acute changes on a minute-based time scale. These data expand the current knowledge of acute, non-transcriptional activities of glucocorticoids, including changes in protein subcellular localization, altered translation of quiescent RNA pools, and PKC-mediated cytoskeleton remodeling.

  10. Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

    Jillian L Blatti

    Full Text Available Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP and thioesterase (TE govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

  11. Optimization and evaluation of surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry for protein profiling of cerebrospinal fluid

    Gomez-Mancilla Baltazar

    2006-04-01

    Full Text Available Abstract Cerebrospinal fluid (CSF potentially carries an archive of peptides and small proteins relevant to pathological processes in the central nervous system (CNS and surrounding brain tissue. Proteomics is especially well suited for the discovery of biomarkers of diagnostic potential in CSF for early diagnosis and discrimination of several neurodegenerative diseases. ProteinChip surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS is one such approach which offers a unique platform for high throughput profiling of peptides and small proteins in CSF. In this study, we evaluated methodologies for the retention of CSF proteins m/z we found a high degree of overlap between the tested array surfaces. The combination of CM10 and IMAC30 arrays was sufficient to represent between 80–90% of all assigned peaks when using either sinapinic acid or α-Cyano-4-hydroxycinnamic acid as the energy absorbing matrices. Moreover, arrays processed with SPA consistently showed better peak resolution and higher peak number across all surfaces within the measured mass range. We intend to use CM10 and IMAC30 arrays prepared in sinapinic acid as a fast and cost-effective approach to drive decisions on sample selection prior to more in-depth discovery of diagnostic biomarkers in CSF using alternative but complementary proteomic strategies.

  12. Redox Proteomic Profiling of Specifically Carbonylated Proteins in the Serum of Triple Transgenic Alzheimer’s Disease Mice

    Liming Shen

    2016-04-01

    Full Text Available Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer’s disease (AD. To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice and the age- and sex-matched non-transgenic (non-Tg littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot, was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin Ig gamma-2B chain C region (IGH-3, Ig lambda-2 chain C region (IGLC2, Ig kappa chain C region (IGKC, and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.

  13. Multi-method research strategy for understanding changes in storage protein composition in developing barley grain to improve nutritional profile

    Kaczmarczyk, Agnieszka Ewa

    Barley (Hordeum vulgare L.) is cultivated in a range of diverse environments and is widely utilised as feed for animal and as malt in brewing. Nitrogen (N) is a key macronutrient whch directly increases plant growth and is used as a fertiliser to meet the demands for higher yield. However, the in...... regimes. To reach the objective, integrated transcriptomics and proteomics analysis complemented with AAs profiling have been undertaken....

  14. Profiling of bacterial cells and cell-surface proteins of plant-associated bacteria by standard analytical techniques

    Moravcová, Dana; Horká, Marie; Šalplachta, Jiří; Kubesová, Anna; Vykydalová, Marie; Kahle, Vladislav

    Latvia: Latvian Institute of Organic Synthesis, 2011 - (Kažoka, H.). s. 94 [Nordic Separation Science Society Conference /6./. 24.09.2011-27.09.2011, Riga] R&D Projects: GA AV ČR IAAX00310701; GA MV VG20112015021 Institutional research plan: CEZ:AV0Z40310501 Keywords : bacterial profiling * Rhizobium * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation http://www.nosss.eu

  15. iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening

    Martínez-Esteso, María José; Vilella-Antón, María Teresa; Pedreño, María Ángeles; Valero, María Luz; Bru-Martínez, Roque

    2013-01-01

    Background: Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components such as sugars, acids, flavors, anthocyanins, tannins, etc., accumulate in the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance in our understanding of berry development and ripening processes. Results: We report the developmental analysis...

  16. Monte Carlo study of thermal flux profiles and body correction factors for body protein measurements of obese subjects

    In previous calculations for total body nitrogen measurements of children, the anterior/posterior thermal neutron flux profile with depth was found to be fairly flat after an initial rise. However, for obese adults significant variations are found in the flux profile with the central flux value being as low as 20% of the peak value. The significance of these flux variations is examined. Correction factors are calculated for the varying attenuation of the nitrogen and hydrogen photons by a range of obese bodies. The calculations included the effect of the thermal flux profile as well as that of an outer layer of low nitrogen content adipose tissue. The bodies are assumed to have a homogeneous hydrogen content. A study of four obese body models with varying sex and fat content shows that the correction factors do not vary much between males and females. This is surprising since the female models are assumed to have a surface fat layer twice as thick as for the male models. The correction factors are found to be only slightly sensitive to the thermal flux variations with depth. 5 refs., 1 tab., 4 figs

  17. Blood profile of proteins and steroid hormones predicts weight change after weight loss with interactions of dietary protein level and glycemic index.

    Ping Wang

    Full Text Available BACKGROUND: Weight regain after weight loss is common. In the Diogenes dietary intervention study, high protein and low glycemic index (GI diet improved weight maintenance. OBJECTIVE: To identify blood predictors for weight change after weight loss following the dietary intervention within the Diogenes study. DESIGN: Blood samples were collected at baseline and after 8-week low caloric diet-induced weight loss from 48 women who continued to lose weight and 48 women who regained weight during subsequent 6-month dietary intervention period with 4 diets varying in protein and GI levels. Thirty-one proteins and 3 steroid hormones were measured. RESULTS: Angiotensin I converting enzyme (ACE was the most important predictor. Its greater reduction during the 8-week weight loss was related to continued weight loss during the subsequent 6 months, identified by both Logistic Regression and Random Forests analyses. The prediction power of ACE was influenced by immunoproteins, particularly fibrinogen. Leptin, luteinizing hormone and some immunoproteins showed interactions with dietary protein level, while interleukin 8 showed interaction with GI level on the prediction of weight maintenance. A predictor panel of 15 variables enabled an optimal classification by Random Forests with an error rate of 24±1%. A logistic regression model with independent variables from 9 blood analytes had a prediction accuracy of 92%. CONCLUSIONS: A selected panel of blood proteins/steroids can predict the weight change after weight loss. ACE may play an important role in weight maintenance. The interactions of blood factors with dietary components are important for personalized dietary advice after weight loss. REGISTRATION: ClinicalTrials.gov NCT00390637.

  18. Activity Based Costing and Product Pricing Decision: the Nigerian Case

    Ebipanipre Gabriel Mieseigha

    2014-01-01

    This paper examined activity based costing and product pricing decisions in Nigeria so as to ascertain whether activity based costing have the ability to enhance profitability and control cost of manufacturing firms. Towards this end, a multiple correlation and regression estimation technique was used in analyzing the data obtained in the study. The study found that activity based costing affects product costing and pricing decision. In addition, the results showed that improved profitability...

  19. Activity based costing at the Naval Postgraduate School

    Belgum, Stephen A.

    1995-01-01

    This thesis uses Activity Based Costing to develop a budgeting model for an academic department at the Naval Postgraduate School. The purpose of this Activity Based Costing model is to provide managers with a more effective means of justifying resources and to function as a budgeting tool. The model consists of three levels: resources, activities, and outputs. The model is a flexible tool that uses an activity based software package. This thesis demonstrates that the model tracks the processe...

  20. Real-time collaboration in activity-based architectures

    Bardram, Jakob Eyvind; Christensen, Henrik Bærbak

    2004-01-01

    With the growing research into mobile and ubiquitous computing, there is a need for addressing how such infrastructures can support collaboration between nomadic users. We present the activity based computing paradigm and outline a proposal for handling collaboration in an activity-based architec......-based architecture. We argue that activity-based computing establishes a natural and sound conceptual and architectural basis for session management in real-time, synchronous collaboration....

  1. The Development of an Angiogenic Protein “Signature” in Ovarian Cancer Ascites as a Tool for Biologic and Prognostic Profiling

    Trachana, Sofia-Paraskevi; Pilalis, Eleftherios; Gavalas, Nikos G.; Tzannis, Kimon; Papadodima, Olga; Liontos, Michalis; Rodolakis, Alexandros; Vlachos, Georgios; Thomakos, Nikolaos; Haidopoulos, Dimitrios; Lykka, Maria; Koutsoukos, Konstantinos; Kostouros, Efthimios; Terpos, Evagelos; Chatziioannou, Aristotelis; Dimopoulos, Meletios-Athanasios; Bamias, Aristotelis

    2016-01-01

    Advanced ovarian cancer (AOC) is one of the leading lethal gynecological cancers in developed countries. Based on the important role of angiogenesis in ovarian cancer oncogenesis and expansion, we hypothesized that the development of an “angiogenic signature” might be helpful in prediction of prognosis and efficacy of anti-angiogenic therapies in this disease. Sixty-nine samples of ascitic fluid- 35 from platinum sensitive and 34 from platinum resistant patients managed with cytoreductive surgery and 1st-line carboplatin-based chemotherapy- were analyzed using the Proteome ProfilerTM Human Angiogenesis Array Kit, screening for the presence of 55 soluble angiogenesis-related factors. A protein profile based on the expression of a subset of 25 factors could accurately separate resistant from sensitive patients with a success rate of approximately 90%. The protein profile corresponding to the “sensitive” subset was associated with significantly longer PFS (8 [95% Confidence Interval {CI}: 8–9] vs. 20 months [95% CI: 15–28]; Hazard ratio {HR}: 8.3, p<0.001) and OS (20.5 months [95% CI: 13.5–30] vs. 74 months [95% CI: 36-not reached]; HR: 5.6 [95% CI: 2.8–11.2]; p<0.001). This prognostic performance was superior to that of stage, histology and residual disease after cytoreductive surgery and the levels of vascular endothelial growth factor (VEGF) in ascites. In conclusion, we developed an “angiogenic signature” for patients with AOC, which can be used, after appropriate validation, as a prognostic marker and a tool for selection for anti-angiogenic therapies. PMID:27258020

  2. Protein profiles and organoleptic properties of bread from wheat flour and full-fat or defatted fermented cocoa bean powder.

    Aremu, C Y; Agiang, M A; Ayatse, J O

    1995-12-01

    This study has shown that the protein in bread may be quantitatively increased significantly by addition of full-fat or defatted cocoa powder to white flour. The recipe in which white flour is incorporated with up to 10 percent defatted cocoa powder gives bread that is nearly as well accepted as white bread, but with a significantly higher protein content than the latter. However, organoleptic acceptability drops with increasing percentage of cocoa supplementation. The bitter taste of theobromine, which is normally present in high amounts in cocoa bean, is thought to be responsible for this problem of poor acceptability of high cocoa breads. This problem will have to be addressed in order to enhance the scope of increasing bread protein by cocoa supplementation. PMID:8882367

  3. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

    Li, Renfeng; Liao, Gangling; Nirujogi, Raja Sekhar; Pinto, Sneha M; Shaw, Patrick G; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S; Pandey, Akhilesh; Hayward, S Diane

    2015-12-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  4. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.

    Feng Yang

    Full Text Available BACKGROUND: High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. PRINCIPAL FINDINGS: We have identified 7117 unique phosphopeptides (2566 phosphoproteins from control and irradiated (2 and 50 cGy primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. CONCLUSIONS: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.

  5. Influence of sinomenine on protein profiles of peripheral blood mononuclear cells from ankylosing spondylitis patients: a pharmacoproteomics study

    HUANG Zhi-xiang; TAN Jin-hui; LI Tian-wang; DENG Wei-ming; QIU Ke-wei; LIAO Ze-tao; ZENG Zhao-qiu

    2013-01-01

    Background Ankylosing spondylitis (AS) is a common inflammatory rheumatic disease which lacks satisfactory treatment so far.Sinomenine (SIN) is an alkaloid and has recently been utilized in treating multiple rheumatic diseases including AS in China,but its exact mechanism remains to be explored.This study investigated the alteration of proteome in peripheral blood mononuclear cells (PBMCs) from AS patients.Methods Thirty AS patients were enrolled in this study.PBMCs from each AS patient were cultured in medium with or without SIN respectively.Then PBMCs proteins from both groups were separated by two-dimensional electrophoresis (2-DE) and analyzed by mass spectrometry (MS).Two differentially expressed proteins were then chosen to be verified using Western blotting.Results Seven proteins,including α-synuclein (SNCA),calmodulin (CALM),acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A),chloride intracellular channel protein 1 (CLIC1),guanine nucleotide-binding protein G(I)/G(S)/ G(T) subunit beta-1 (GNB1),gelsolin (GSN) and histone H2B type 1-M (HISTH2BM)were over-expressed,while coronin1A (CORO1A) was under-expressed in the SIN-treated PBMCs.Further bioinformatics search indicated that the changes of SNCA,ANP32A and CLIC1 pertained to apoptosis,while changes of GSN and CORO1A were associated with both apoptosis and inhibition of immunological function.Subsequently GSN and CORO1A were selected to validate by Western blotting and the results were consistent with those of 2-DE.Conclusion There were 8 differentially expressed proteins in the SIN-treated PBMCs,which might shed some light on the mechanism of SIN in the treatment of AS.

  6. A potential new selection criterion for breeding winter barley optimal protein and amino acid profiles for liquid pig feed

    Christensen, Jesper Bjerg; Blaabjerg, Karoline; Poulsen, Hanne Damgaard

    concentration was analysed in the supernatant after centrifugation. After 15 min., app. 16% of the total protein was soluble and until 8 hours an increase of 5% units was observed. However, from 8 to 48 hours it increased with 10% units for some cultivars. Based on these analyses, cultivars were selected for...... selection criterion for barley to be used in feeding....

  7. Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles

    Antroquinonol a derivative of Antrodia camphorata has been reported to have antitumor effects against various cancer cells. However, the effect of antroquinonol on cell signalling and survival pathways in non-small cell lung cancer (NSCLC) cells has not been fully demarcated. Here we report that antroquinonol treatment significantly reduced the proliferation of three NSCLC cells. Treatment of A549 cells with antroquinonol increased cell shrinkage, apoptotic vacuoles, pore formation, TUNEL positive cells and increased Sub-G1 cell population with respect to time and dose dependent manner. Antroquinonol treatment not only increased the Sub-G1 accumulation but also reduced the protein levels of cdc2 without altering the expression of cyclin B1, cdc25C, pcdc2, and pcdc25C. Antroquinonol induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of Caspase 3 and PARP cleavage in A549 cells. Moreover, antroquinonol treatment down regulated the expression of Bcl2 proteins, which was correlated with the decreased PI3K and mTOR protein levels without altering pro apoptotic and anti apoptotic proteins. Results from the microarray analysis demonstrated that antroquinonol altered the expression level of miRNAs compared with untreated control in A549 cells. The data collectively suggested the antiproliferative effect of antroquinonol on NSCLC A549 cells, which provides useful information for understanding the anticancer mechanism influenced by antroquinonol and is the first report to suggest that antroquinonol may be a promising chemotherapeutic agent for lung cancer.

  8. Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles.

    Kumar, V Bharath; Yuan, Ta-Chun; Liou, Je-Wen; Yang, Chih-Jen; Sung, Ping-Jyun; Weng, Ching-Feng

    2011-02-10

    Antroquinonol a derivative of Antrodia camphorata has been reported to have antitumor effects against various cancer cells. However, the effect of antroquinonol on cell signalling and survival pathways in non-small cell lung cancer (NSCLC) cells has not been fully demarcated. Here we report that antroquinonol treatment significantly reduced the proliferation of three NSCLC cells. Treatment of A549 cells with antroquinonol increased cell shrinkage, apoptotic vacuoles, pore formation, TUNEL positive cells and increased Sub-G1 cell population with respect to time and dose dependent manner. Antroquinonol treatment not only increased the Sub-G1 accumulation but also reduced the protein levels of cdc2 without altering the expression of cyclin B1, cdc25C, pcdc2, and pcdc25C. Antroquinonol induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of Caspase 3 and PARP cleavage in A549 cells. Moreover, antroquinonol treatment down regulated the expression of Bcl2 proteins, which was correlated with the decreased PI3K and mTOR protein levels without altering pro apoptotic and anti apoptotic proteins. Results from the microarray analysis demonstrated that antroquinonol altered the expression level of miRNAs compared with untreated control in A549 cells. The data collectively suggested the antiproliferative effect of antroquinonol on NSCLC A549 cells, which provides useful information for understanding the anticancer mechanism influenced by antroquinonol and is the first report to suggest that antroquinonol may be a promising chemotherapeutic agent for lung cancer. PMID:21185843

  9. Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles

    Kumar, V. Bharath; Yuan, Ta-Chun [Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan (China); Liou, Je-Wen [Department of Biochemistry, School of Medicine, Tzu-Chi University, Hualien, Taiwan (China); Yang, Chih-Jen [Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan (China); Sung, Ping-Jyun [Graduate Institute of Marine Biotechnology, Department of Life Science, National Dong Hwa University, Pingtung, Taiwan (China); Weng, Ching-Feng, E-mail: cfweng@mail.ndhu.edu.tw [Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan (China)

    2011-02-10

    Antroquinonol a derivative of Antrodia camphorata has been reported to have antitumor effects against various cancer cells. However, the effect of antroquinonol on cell signalling and survival pathways in non-small cell lung cancer (NSCLC) cells has not been fully demarcated. Here we report that antroquinonol treatment significantly reduced the proliferation of three NSCLC cells. Treatment of A549 cells with antroquinonol increased cell shrinkage, apoptotic vacuoles, pore formation, TUNEL positive cells and increased Sub-G1 cell population with respect to time and dose dependent manner. Antroquinonol treatment not only increased the Sub-G1 accumulation but also reduced the protein levels of cdc2 without altering the expression of cyclin B1, cdc25C, pcdc2, and pcdc25C. Antroquinonol induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of Caspase 3 and PARP cleavage in A549 cells. Moreover, antroquinonol treatment down regulated the expression of Bcl2 proteins, which was correlated with the decreased PI3K and mTOR protein levels without altering pro apoptotic and anti apoptotic proteins. Results from the microarray analysis demonstrated that antroquinonol altered the expression level of miRNAs compared with untreated control in A549 cells. The data collectively suggested the antiproliferative effect of antroquinonol on NSCLC A549 cells, which provides useful information for understanding the anticancer mechanism influenced by antroquinonol and is the first report to suggest that antroquinonol may be a promising chemotherapeutic agent for lung cancer.

  10. Integrated transcriptomics and proteomics analysis of storage protein composition in developing barley grain to improve nutritional profile

    Kaczmarczyk, Agnieszka Ewa; Dionisio, Giuseppe; Renaut, Jenny;

    2012-01-01

    The aim of the study was to understand the molecular and biochemical mechanisms underpinning the effect of nitrogen (N) on barley (Hordeum vulgare) storage protein production (hordeins) during grain filling. Using a combination of advanced biochemistry methods, we could comprehensively describe...

  11. Molecular Profiling of Synaptic Vesicle Docking Sites Reveals Novel Proteins but Few Differences between Glutamatergic and GABAergic Synapses

    Boyken, Janina; Gronborg, Mads; Riedel, Dietmar; Urlaub, Henning; Jahn, Reinhard; Chua, John Jia En

    2013-01-01

    Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to

  12. Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach.

    Broncel, Malgorzata; Serwa, Remigiusz A; Bunney, Tom D; Katan, Matilda; Tate, Edward W

    2016-02-01

    AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation. PMID:26604261

  13. Alterations in the fatty acid profile, antioxidant enzymes and protein pattern of Biomphalaria alexandrina snails exposed to the pesticides diazinon and profenfos.

    Bakry, Fayez A; El-Hommossany, Karem; Abd El-Atti, Mahmoud; Ismail, Somaya M

    2016-04-01

    The use of pesticides is widespread in agricultural activities. These pesticides may contaminate the irrigation and drainage systems during agriculture activities and pests' control and then negatively affect the biotic and a biotic component of the polluted water courses. The present study aimed to evaluate the effect of the pesticides diazinon and profenfos on some biological activities of Biomphalaria alexandrina snails such as fatty acid profile, some antioxidant enzymes (thioredoxin reductase (TrxR), sorbitol dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT) as well as glutathione reductase (GR) and lipid peroxidation (LP)) and protein patterns in snails' tissues exposed for 4 weeks to LC10 of diazinon and profenfos. The results showed that the two pesticides caused considerable reduction in survival rates and egg production of treated snails. Identification of fatty acid composition in snail tissues treated with diazinon and profenfos pesticides was carried out using gas-liquid chromatography (GLC). The results declared alteration in fatty acid profile, fluctuation in percentage of long chain and short chain fatty acid contributions either saturated or unsaturated ones, and a decrease in total lipid content in tissues of snails treated with these pesticides. The data demonstrate that there was a significant inhibition in the activities of tissues SOD, CAT, glutathione reductase (GR), TrxR, and SDH in tissues of treated snails, while a significant elevation was detected in LP as compared to the normal control. On the other hand, the electrophoretic pattern of total protein showed differences in number and molecular weights of protein bands due to the treatment of snails. It was concluded that the residues of diazinon and profenfos pesticides in aquatic environments have toxic effects onB. alexandrina snails. PMID:24215063

  14. Two novel LRR-only proteins in Chlamys farreri: Similar in structure, yet different in expression profile and pattern recognition.

    Wang, Mengqiang; Wang, Lingling; Xin, Lusheng; Wang, Xiudan; Wang, Lin; Xu, Jianchao; Jia, Zhihao; Yue, Feng; Wang, Hao; Song, Linsheng

    2016-06-01

    Leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and be involved in the immune response. In the present study, two novel LRR-only proteins, CfLRRop-2 and CfLRRop-3, were identified and characterized from scallop Chlamys farreri. They both contained nine LRR motifs with the consensus signature sequence LxxLxLxxNxL and formed typical horseshoe structure. The CfLRRop-2 and CfLRRop-3 mRNA transcripts were constitutively expressed in haemocytes, muscle, mantle, gill, haepatopancreas and gonad, with the highest expression level in haepatopancreas and gill, respectively. During the ontogenesis of scallop, the mRNA transcripts of CfLRRop-2 were kept at a high level in oocytes and embryos, while those of CfLRRop-3 were expressed at a rather low level from oocytes to blastula. Their mRNA transcripts were significantly increased after the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), and the mRNA expression of CfLRRop-2 rose more intensely than that of CfLRRop-3. After the suppression of CfTLR (previously identified Toll-like receptor in C. farreri) via RNA interference (RNAi), CfLRRop-3 mRNA transcripts increased more intensely and lastingly than those of CfLRRop-2. The rCfLRRop-3 protein could bind LPS, PGN, GLU and poly I:C, while rCfLRRop-2 exhibited no significant binding activity to them. Additionally, rCfLRRop-2 could significantly induce the release of TNF-α from the mixed primary cultured scallop haemocytes, but rCfLRRop-3 failed. These results collectively indicated that CfLRRop-2 might act as an immune effector or pro-inflammatory factor, while CfLRRop-3 would function as a pattern recognition receptor (PRR), suggesting the function of LRR-only protein family has differentiated in scallop. PMID:26826425

  15. The Effect of Feeding with Different Dietary Protein Levels on Haematological Profile and Leukocytes Population of Juvenile Paddlefish, Polyodon spathula

    Marian Tiberiu Coadă

    2012-10-01

    Full Text Available The aim of this paper is to evaluate the haematological parameters and the leukogram of paddlefish juveniles fed with different protein level diets. The experiment was conducted in a recirculating system, populated with 112 days old paddlefish juveniles with a mean weight of 57.93 g. During the experiment, two different types of pellets were tested: Aller Performa (54% crude protein at variant V1 (B1, B3 and Classic Extra 1P (41% crude protein at variant V2 (B2,B4. The haematological parameters analyzed were: RBC, Hb, Ht, MCV, MCH and MCHC. In V1 case, RBC, Hb, Ht and MCV registered lower values than those from V2 while MCH and MCHC were higher. By study all the different types of leukocyte found on blood smears (lymphocytes, monocytes and granulocytes, the leukogram was determined. The physiological stress induced by different protein level diets reflected the absolute number of leukocytes. Thus, at V2, the value of 82.05 x 103 WBC/mm3blood was significantly higher than the one from V1 (50.80 x 103 WBC/mm3blood. A decrease of lymphocytes percentage was observed in case of V1 (59%, comparing to V2 (71.125%. This shows a decrease of fish immunity, but in exchange, the percentage of neutrophils increased. As a conclusion, in terms of physiological condition, percentage of 41% crude protein (V2 led to a better health condition of paddlefish juveniles

  16. Quantitative Profiling Identifies Potential Regulatory Proteins Involved in Development from Dauer Stage to L4 Stage in Caenorhabditis elegans.

    Kim, Sunhee; Lee, Hyoung-Joo; Hahm, Jeong-Hoon; Jeong, Seul-Ki; Park, Don-Ha; Hancock, William S; Paik, Young-Ki

    2016-02-01

    When Caenorhabditis elegans encounters unfavorable growth conditions, it enters the dauer stage, an alternative L3 developmental period. A dauer larva resumes larval development to the normal L4 stage by uncharacterized postdauer reprogramming (PDR) when growth conditions become more favorable. During this transition period, certain heterochronic genes involved in controlling the proper sequence of developmental events are known to act, with their mutations suppressing the Muv (multivulva) phenotype in C. elegans. To identify the specific proteins in which the Muv phenotype is highly suppressed, quantitative proteomic analysis with iTRAQ labeling of samples obtained from worms at L1 + 30 h (for continuous development [CD]) and dauer recovery +3 h (for postdauer development [PD]) was carried out to detect changes in protein abundance in the CD and PD states of both N2 and lin-28(n719). Of the 1661 unique proteins identified with a < 1% false discovery rate at the peptide level, we selected 58 proteins exhibiting ≥2-fold up-regulation or ≥2-fold down-regulation in the PD state and analyzed the Gene Ontology terms. RNAi assays against 15 selected up-regulated genes showed that seven genes were predicted to be involved in higher Muv phenotype (p < 0.05) in lin-28(n791), which is not seen in N2. Specifically, two genes, K08H10.1 and W05H9.1, displayed not only the highest rate (%) of Muv phenotype in the RNAi assay but also the dauer-specific mRNA expression, indicating that these genes may be required for PDR, leading to the very early onset of dauer recovery. Thus, our proteomic approach identifies and quantitates the regulatory proteins potentially involved in PDR in C. elegans, which safeguards the overall lifecycle in response to environmental changes. PMID:26751275

  17. Chia Seed Shows Good Protein Quality, Hypoglycemic Effect and Improves the Lipid Profile and Liver and Intestinal Morphology of Wistar Rats.

    da Silva, Bárbara Pereira; Dias, Desirrê Morais; de Castro Moreira, Maria Eliza; Toledo, Renata Celi Lopes; da Matta, Sérgio Luis Pinto; Lucia, Ceres Mattos Della; Martino, Hércia Stampini Duarte; Pinheiro-Sant'Ana, Helena Maria

    2016-09-01

    Chia has been consumed by the world population due to its high fiber, lipids and proteins content. The objective was to evaluate the protein quality of chia untreated (seed and flour) and heat treated (90 °C/20 min), their influence on glucose and lipid homeostasis and integrity of liver and intestinal morphology of Wistar rats. 36 male rats, weanling, divided into six groups which received control diet (casein), free protein diet (aproteic) and four diet tests (chia seed; chia seed with heat treatment; chia flour and chia flour with heat treatment) for 14 days were used. The protein efficiency ratio (PER), net protein ratio (NPR) and true digestibility (TD) were evaluated. The biochemical variables and liver and intestinal morphologies of animals were determined. The values of PER, NPR and TD did not differ among the animals that were fed with chia and were lower than the control group. The animals that were fed with chia showed lower concentrations of glucose; triacylglycerides, low-density lipoprotein cholesterol and very low-density lipoprotein and higher high-density lipoprotein cholesterol than the control group. The liver weight of animals that were fed with chia was lower than the control group. Crypt depth and thickness of intestinal muscle layers were higher in groups that were fed with chia. The consumption of chia has shown good digestibility, hypoglycemic effect, improved lipid and glycemic profiles and reduced fat deposition in liver of animals, and also promoted changes in intestinal tissue that enhanced its functionality. PMID:27193017

  18. Operating cost analysis of anaesthesia: Activity based costing (ABC analysis

    Majstorović Branislava M.

    2011-01-01

    Full Text Available Introduction. Cost of anaesthesiology represent defined measures to determine a precise profile of expenditure estimation of surgical treatment, which is important regarding planning of healthcare activities, prices and budget. Objective. In order to determine the actual value of anaestesiological services, we started with the analysis of activity based costing (ABC analysis. Methods. Retrospectively, in 2005 and 2006, we estimated the direct costs of anestesiological services (salaries, drugs, supplying materials and other: analyses and equipment. of the Institute of Anaesthesia and Resuscitation of the Clinical Centre of Serbia. The group included all anesthetized patients of both sexes and all ages. We compared direct costs with direct expenditure, “each cost object (service or unit” of the Republican Health-care Insurance. The Summary data of the Departments of Anaesthesia documented in the database of the Clinical Centre of Serbia. Numerical data were utilized and the numerical data were estimated and analyzed by computer programs Microsoft Office Excel 2003 and SPSS for Windows. We compared using the linear model of direct costs and unit costs of anaesthesiological services from the Costs List of the Republican Health-care Insurance. Results. Direct costs showed 40% of costs were spent on salaries, (32% on drugs and supplies, and 28% on other costs, such as analyses and equipment. The correlation of the direct costs of anaestesiological services showed a linear correlation with the unit costs of the Republican Healthcare Insurance. Conclusion. During surgery, costs of anaesthesia would increase by 10% the surgical treatment cost of patients. Regarding the actual costs of drugs and supplies, we do not see any possibility of costs reduction. Fixed elements of direct costs provide the possibility of rationalization of resources in anaesthesia.

  19. Activity Based Startup Plan for Prototype Vertical Denitration Calciner

    Testing activities on the Prototype Vertical Denitration Calciner at PFP were suspended in January 1997 due to the hold on fissile material handling in the facility. The restart of testing activities will require a review through an activity based startup process based upon Integrated Safety Management (ISM) principles to verify readiness. The Activity Based Startup Plan has been developed for this process

  20. Dashboard auditing of ABC (Activity-Based Costing). Theoretical approaches

    Căpuşneanu, Sorinel/I

    2009-01-01

    This article aims to define the dashboard auditing according to the specifics of Activity-Based Costing method (ABC). It describes the main objectives of dashboard auditing, the criteria that a dashboard auditor should meet and the step-by-step stages of the entire dashboard auditing process of an enterprise from steel industry according to the Activity-Based Costing method (ABC).

  1. Dashboard Auditing of Activity-Based Costing (ABC)

    Sorinel Capusneanu

    2009-01-01

    This article aims to define the dashboard auditing according to the specifics of Activity-Based Costing method (ABC). It describes the main objectives of dashboard auditing, the criteria that a dashboard auditor should meet and the step-by-step stages of the entire dashboard auditing process according to the Activity-Based Costing method (ABC).

  2. Dashboard Auditing of Activity-Based Costing (ABC

    Sorinel Capusneanu

    2009-03-01

    Full Text Available This article aims to define the dashboard auditing according to the specifics of Activity-Based Costing method (ABC. It describes the main objectives of dashboard auditing, the criteria that a dashboard auditor should meet and the step-by-step stages of the entire dashboard auditing process according to the Activity-Based Costing method (ABC.

  3. Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

    Verena Natalie Schreier

    Full Text Available Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac-His-Asp-Trp-Lys(Da  = Aoa-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl. This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

  4. Profile of the GSK published protein kinase inhibitor set across ATP-dependent and-independent luciferases: implications for reporter-gene assays.

    Dranchak, Patricia; MacArthur, Ryan; Guha, Rajarshi; Zuercher, William J; Drewry, David H; Auld, Douglas S; Inglese, James

    2013-01-01

    A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses. PMID:23505445

  5. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. PMID:26049080

  6. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen; Mann, Matthias; Jensen, Ole Nørregaard; Kassem, Moustapha

    2005-01-01

    of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and......One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely...... in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral...

  7. Genome-wide identification and analysis of expression profiles of maize mitogen-activated protein kinase kinase kinase.

    Xiangpei Kong

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.

  8. Protein Profiling of Isolated Leukocytes, Myofibroblasts, Epithelial, Basal, and Endothelial Cells from Normal, Hyperplastic, Cancerous, and Inflammatory Human Prostate Tissues

    Zahraa I. Khamis, Kenneth A. Iczkowski, Ziad J. Sahab, Qing-Xiang Amy Sang

    2010-01-01

    Full Text Available In situ neoplastic prostate cells are not lethal unless they become invasive and metastatic. For cells to become invasive, the prostate gland must undergo degradation of the basement membrane and disruption of the basal cell layer underneath the luminal epithelia. Although the roles of proteinases in breaking down the basement membrane have been well-studied, little is known about the factors that induce basal cell layer disruption, degeneration, and its eventual disappearance in invasive cancer. It is hypothesized that microenvironmental factors may affect the degradation of the basal cell layer, which if protected may prevent tumor progression and invasion. In this study, we have revealed differential protein expression patterns between epithelial and stromal cells isolated from different prostate pathologies and identified several important epithelial and stromal proteins that may contribute to inflammation and malignant transformation of human benign prostate tissues to cancerous tissues using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and proteomics methods. Cellular retinoic acid-binding protein 2 was downregulated in basal cells of benign prsotate. Caspase-1 and interleukin-18 receptor 1 were highly expressed in leukocytes of prostate cancer. Proto-oncogene Wnt-3 was downregulated in endothelial cells of prostatitis tissue and tyrosine phosphatase non receptor type 1 was only found in normal and benign endothelial cells. Poly ADP-ribose polymerase 14 was downregulated in myofibroblasts of prostatitis tissue. Interestingly, integrin alpha-6 was upregulated in epithelial cells but not detected in myofibroblasts of prostate cancer. Further validation of these proteins may generate new strategies for the prevention of basal cell layer disruption and subsequent cancer invasion.

  9. Plasma protein profiling of Mild Cognitive Impairment and Alzheimer’s disease using iTRAQ quantitative proteomics

    Song, Fei; Poljak, Anne; Nicole A Kochan; Raftery, Mark; Brodaty, Henry; Smythe, George A.; Perminder S Sachdev

    2014-01-01

    Background With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline. Methods Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 ...

  10. The Impact of Tissue Fixatives on Morphology and Antibody-based Protein Profiling in Tissues and Cells

    Paavilainen, Linda; Edvinsson, Åsa; Asplund, Anna; Hober, Sophia; Kampf, Caroline; Pontén, Fredrik; Wester, Kenneth

    2010-01-01

    Pathology archives harbor large amounts of formalin-fixed, paraffin-embedded tissue samples, used mainly in clinical diagnostics but also for research purposes. Introduction of heat-induced antigen retrieval has enabled the use of tissue samples for extensive immunohistochemical analysis, despite the fact that antigen retrieval may not recover all epitopes, owing to alterations of the native protein structure induced by formalin. The aim of this study was to investigate how different fixative...

  11. Expression profile of plakin cross-linking proteins in short-term denervated mouse hindlimb skeletal muscle

    Blouin, Patrick; Serresse,Olivier; Dorman,Sandra; Larivière,Céline

    2016-01-01

    Patrick Blouin,1 Olivier Serresse,1 Sandra C Dorman,1,2 Céline Larivière,1–3 1School of Human Kinetics, 2Northern Ontario School of Medicine, 3Biomolecular Sciences, Laurentian University, Sudbury, ON, Canada Purpose: Skeletal muscle atrophy linked to neuromuscular inactivity is a complex phenomenon involving widespread alteration of muscle structure and function. Plakin cross-linking proteins are important structural elements that are expressed in skeletal mus...

  12. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples

    Belov, Larissa; Matic, Kieran J.; Hallal, Susannah; Mulligan, Stephen P.; Best, O. Giles; Christopherson, Richard I

    2016-01-01

    Extracellular vesicles (EV) are membranous particles (30–1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surfa...

  13. Biotype Characterization, Developmental Profiling, Insecticide Response and Binding Property of Bemisia tabaci Chemosensory Proteins: Role of CSP in Insect Defense.

    Guoxia Liu

    Full Text Available Chemosensory proteins (CSPs are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1 was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. α-pentyl-cinnamaldehyde (jasminaldehyde. This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity.

  14. Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

    Currie, Devin [Dartmouth College; Guss, Adam M [ORNL; Herring, Christopher [Mascoma Corporation; Giannone, Richard J [ORNL; Johnson, Courtney M [ORNL; Lankford, Patricia K [ORNL; Brown, Steven D [ORNL; Hettich, Robert {Bob} L [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2014-01-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars

  15. Biotype Characterization, Developmental Profiling, Insecticide Response and Binding Property of Bemisia tabaci Chemosensory Proteins: Role of CSP in Insect Defense

    Liu, Guoxia; Ma, Hongmei; Xie, Hongyan; Xuan, Ning; Guo, Xia; Fan, Zhongxue; Rajashekar, Balaji; Arnaud, Philippe; Offmann, Bernard; Picimbon, Jean-François

    2016-01-01

    Chemosensory proteins (CSPs) are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1) was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. α-pentyl-cinnamaldehyde (jasminaldehyde). This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity. PMID:27167733

  16. Biotype Characterization, Developmental Profiling, Insecticide Response and Binding Property of Bemisia tabaci Chemosensory Proteins: Role of CSP in Insect Defense.

    Liu, Guoxia; Ma, Hongmei; Xie, Hongyan; Xuan, Ning; Guo, Xia; Fan, Zhongxue; Rajashekar, Balaji; Arnaud, Philippe; Offmann, Bernard; Picimbon, Jean-François

    2016-01-01

    Chemosensory proteins (CSPs) are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1) was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. α-pentyl-cinnamaldehyde (jasminaldehyde). This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity. PMID:27167733

  17. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

    Yansheng Liu

    Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

  18. Identification and expression profile of a putative basement membrane protein gene in the midgut of Helicoverpa armigera

    Xu Da-Wei

    2007-06-01

    Full Text Available Abstract Background The midgut undergoes histolysis and remodeling during the larval to adult transition in holometabolous insects, but the molecular mechanisms underlying this process are not well understood. Results Using Suppression Subtractive Hybridization (SSH, we identified a 531 bp cDNA predicted to encode a 176 amino acid protein, which we call hmg176. Northern and western blot analysis suggested that high levels of hmg176 are expressed in the midgut during molting, but not during metamorphosis. HMG176 protein was detected by immunofluorescence within the membrane of fat bodies and the basement membrane of the midgut of both molting and feeding larvae, but not in metamorphically committed larvae. In situ hybridization revealed that hmg176 transcripts mainly localized to the columnar cells of the midgut. Interestingly, a non-steroidal ecdysone agonist, RH-2485, significantly upregulated expression of hmg176. Conclusion These observations suggest that hmg176 encodes a larval-specific protein that may participate in sustaining larval midgut during larval development, possibly in response to ecdysteroid in vivo. This study will enlighten our understanding of the molecular mechanisms of tissue histolysis during metamorphosis.

  19. Profile of secreted hydrolases, associated proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the degradation of hemicellulose.

    Currie, D H; Guss, A M; Herring, C D; Giannone, R J; Johnson, C M; Lankford, P K; Brown, S D; Hettich, R L; Lynd, L R

    2014-08-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars. PMID:24907337

  20. Proteome profiling of the growth phases of Leishmania pifanoi promastigotes in axenic culture reveals differential abundance of immunostimulatory proteins.

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Mena, María del Carmen; Ciordia, Sergio; Larraga, Vicente

    2016-06-01

    Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the "Leishmania mexicana complex" mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the "L. mexicana complex" allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi. PMID:26992294