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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. 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...

  7. 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...

  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. 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)

  11. 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

  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. 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 ...

  17. 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...

  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. 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 ...

  2. 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....

  3. 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.

  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

    2016-03-01

    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

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

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-15

    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

  5. 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.

  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. 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.

  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. 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.

  10. 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....

  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. 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

  16. 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

  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. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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.

  16. 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

  17. 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

  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. 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

  20. 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.

  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. 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 ...

  18. 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

  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

    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

  2. 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.

  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. 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.

  5. 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.

  6. 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

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  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. 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.

  19. 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

  20. 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

  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. 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...

  9. 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

  10. 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 ...

  11. 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

  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. 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.

  1. 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.

  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. 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...

  4. 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...

  5. 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.

  6. 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.

  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. 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

  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. 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....

  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. 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...

  3. 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...

  4. 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...

  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. 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...

  8. 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.

  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. ''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.)

  17. 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

  18. 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

  19. 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

  20. 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

  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.