Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

Science.gov (United States)

Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

Science.gov (United States)

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

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

Science.gov (United States)

Ismail, Hanafy M; Barton, Victoria E; Panchana, Matthew; Charoensutthivarakul, Sitthivut; Biagini, Giancarlo A; Ward, Stephen A; O'Neill, Paul M

2016-05-23

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.

Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

Science.gov (United States)

Galinski, Sabrina; Wichert, Sven P; Rossner, Moritz J; Wehr, Michael C

2018-05-25

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a β-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.

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

International Nuclear Information System (INIS)

Gao, Wei-Min; Haab, Brian B; Hanash, Samir M; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S

2005-01-01

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

Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

Science.gov (United States)

Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

2013-01-01

Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

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

Science.gov (United States)

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

2015-10-23

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.

HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts.

Science.gov (United States)

Oberthür, C; Jäggi, R; Hamburger, M

2005-06-01

In the pursuit of the anti-inflammatory constituents in lipophilic woad extracts, the 5-lipoxygenase (5-LOX) inhibitory activity was investigated by HPLC-based activity profiling. In a low-resolution profiling, two time windows with peaks of activity were found. The first coincided with tryptanthrin, a known dual inhibitor of cyclooxygenase-2 (COX-2) and 5-LOX, whereas the major inhibitory fraction was towards the end of the HPLC run. The active fractions were profiled in a peak-resolved manner, and the compounds analyzed by LC-MS, GC and TLC. The activity in the lipophilic fractions of the Isatis extract could be linked to an unsaturated fatty acid, alpha-linolenic acid.

PROFIL PROTEIN SUSU DAN PRODUK OLAHANNYA

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R. Susanti

2017-03-01

Full Text Available Penelitian ini bertujuan untuk menganalisis kadar protein dan profil protein pada beberapa susu (susu kedelai, susu kambing dan olahannya (yogurt, tofu. Kadar protein diukur dengan metode Lowry, sedangkan profil protein dianalisis menggunakan SDS PAGE. Data yang diperoleh dianalisis secara deskriptif. Kadar protein tertinggi pada sampel yang dianalisis terdapat pada produk yogurt A (579,5 mg/ml, disusul susu kedelai (289,99 mg/ml dan susu kambing (133,1 mg/ml. Analisis profil protein terlihat pita protein dengan mobilitas terendah sampai tertinggi terletak pada berat molekul 14-150 KDa. Pita protein khas yang hanya dimiliki susu kambing adalah pita 150kDa. Sementara pita protein khas yang hanya dimiliki susu kedelai adalah pita 44 kDa dan 55kDa. Pita protein yang khas hanya dimiliki yogurt A (dengan bakteri Lactobacillus bulgaricus dan Streptococcus thermophillus adalah pita 65Da. Semua jenis susu dan olahannya memiliki pita 70kDa, kecuali susu kedelai. Profil protein susu kedelai dan tofu menunjukkan profil protein yang sangat berbeda, namun keduanya memiliki pita 18kDa.This study aimed to observe protein level and profiles on some milks (soy milk, goat's milk and dairy (yogurt, tofu product. Protein content was observed by Lowry method, whereas the protein profiles were analyzed by polyacrylamide gel electrophoresis. Data were analyzed descriptively. The highest protein content of the observed sample was in yogurt A products (579,5 mg/ml, followed by soy milk (289,99 mg/ml and goat's milk (133,1 mg/ml. Analysis of protein profiles showed protein bands with lowest to highest mobility lies in the molecular weight of 14-150 KDa. Typical protein band of goat's milk was a 150kDa band. While the typical protein bands of soy milk were 44 kDa and 55kDa band. The typical protein band of yogurt A (with Lactobacillus bulgaricus and Streptococcus thermophillus bacterium was 65Da. All types of milks and dairy had 70kDa band, except for soy milk. Protein

Network-based analysis of proteomic profiles

KAUST Repository

Wong, Limsoon

2016-01-26

Mass spectrometry (MS)-based proteomics is a widely used and powerful tool for profiling systems-wide protein expression changes. It can be applied for various purposes, e.g. biomarker discovery in diseases and study of drug responses. Although RNA-based high-throughput methods have been useful in providing glimpses into the underlying molecular processes, the evidences they provide are indirect. Furthermore, RNA and corresponding protein levels have been known to have poor correlation. On the other hand, MS-based proteomics tend to have consistency issues (poor reproducibility and inter-sample agreement) and coverage issues (inability to detect the entire proteome) that need to be urgently addressed. In this talk, I will discuss how these issues can be addressed by proteomic profile analysis techniques that use biological networks (especially protein complexes) as the biological context. In particular, I will describe several techniques that we have been developing for network-based analysis of proteomics profile. And I will present evidence that these techniques are useful in identifying proteomics-profile analysis results that are more consistent, more reproducible, and more biologically coherent, and that these techniques allow expansion of the detected proteome to uncover and/or discover novel proteins.

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

DEFF Research Database (Denmark)

Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E

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 original...... studies reporting discriminatory protein peaks for breast cancer as either protein identity or as m/ z values in the period from January 1995 to October 2006. To address the important aspect of reproducibility of mass spectrometry data across different clinical studies, we compared the published lists...... of potential discriminatory peaks with those peaks detected in an original MALDI MS protein profiling study performed by our own research group. A total of 20 protein/peptide profiling studies were eligible for inclusion in the systematic review. Only 3 reports included information on protein identity...

Animal and Plant Proteins as Precursors of Peptides with ACE Inhibitory Activity – An in silico Strategy of Protein Evaluation

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Anna Iwaniak

2009-01-01

Full Text Available This paper presents a modern in silico approach useful in the evaluation of proteins as a source of ACE inhibitors. All protein sequences analyzed were derived from the BIOPEP database. To determine the protein value, the following criteria of evaluation were applied: the profile of potential biological (ACE inhibitory activity of a protein, the frequency of the occurrence of fragments with ACE inhibitory activity (A and the potential biological activity of a protein (B. The results, based on a statistical analysis, indicate that milk proteins can be a better source of ACE inhibitors than wheat gliadins. Moreover, all analyzed gliadins possessed more potent ACE inhibitors than chicken meat proteins. No significant differences were observed when comparing A values between soy globulins and β-lactoglobulins. Although criteria such as the profile of potential biological activity of protein, as well as parameters A and B, can be suitable tools in protein evaluation, the proteolytic digestion of protein needs to be considered. Moreover, computerised methods of classifying proteins according to different algorithms are often subjective due to discretion in interpretation of the results.

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

DEFF Research Database (Denmark)

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...... phylogenetic profiles often require very different RT sets to support high prediction accuracy....

Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density.

Science.gov (United States)

Byström, Sanna; Eklund, Martin; Hong, Mun-Gwan; Fredolini, Claudia; Eriksson, Mikael; Czene, Kamila; Hall, Per; Schwenk, Jochen M; Gabrielson, Marike

2018-02-14

Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density. Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI). Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.

Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

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Cuncong Zhong

2016-07-01

Full Text Available 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

Gene expression profiling of low-grade endometrial stromal sarcoma indicates fusion protein-mediated activation of the Wnt signaling pathway.

Science.gov (United States)

Przybyl, Joanna; Kidzinski, Lukasz; Hastie, Trevor; Debiec-Rychter, Maria; Nusse, Roel; van de Rijn, Matt

2018-05-01

Low-grade endometrial stromal sarcomas (LGESS) harbor chromosomal translocations that affect proteins associated with chromatin remodeling Polycomb Repressive Complex 2 (PRC2), including SUZ12, PHF1 and EPC1. Roughly half of LGESS also demonstrate nuclear accumulation of β-catenin, which is a hallmark of Wnt signaling activation. However, the targets affected by the fusion proteins and the role of Wnt signaling in the pathogenesis of these tumors remain largely unknown. Here we report the results of a meta-analysis of three independent gene expression profiling studies on LGESS and immunohistochemical evaluation of nuclear expression of β-catenin and Lef1 in 112 uterine sarcoma specimens obtained from 20 LGESS and 89 LMS patients. Our results demonstrate that 143 out of 310 genes overexpressed in LGESS are known to be directly regulated by SUZ12. In addition, our gene expression meta-analysis shows activation of multiple genes implicated in Wnt signaling. We further emphasize the role of the Wnt signaling pathway by demonstrating concordant nuclear expression of β-catenin and Lef1 in 7/16 LGESS. Based on our findings, we suggest that LGESS-specific fusion proteins disrupt the repressive function of the PRC2 complex similar to the mechanism seen in synovial sarcoma, where the SS18-SSX fusion proteins disrupt the mSWI/SNF (BAF) chromatin remodeling complex. We propose that these fusion proteins in LGESS contribute to overexpression of Wnt ligands with subsequent activation of Wnt signaling pathway and formation of an active β-catenin/Lef1 transcriptional complex. These observations could lead to novel therapeutic approaches that focus on the Wnt pathway in LGESS. Copyright © 2018 Elsevier Inc. All rights reserved.

The first decade of MALDI protein profiling

DEFF Research Database (Denmark)

Albrethsen, Jakob

2011-01-01

MALDI protein profiling has identified several important challenges in omics-based biomarker research. First, research into the analytical performance of a novel omics-platform of potential diagnostic impact must be carried out in a critical manner and according to common guidelines. Evaluation s...

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

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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.

Proteomic characterization of the human centrosome by protein correlation profiling

DEFF Research Database (Denmark)

Andersen, Jens S; Wilkinson, Christopher J; Mayor, Thibault

2003-01-01

chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human...... centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel...... components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity....

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

DEFF Research Database (Denmark)

Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E

2008-01-01

it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total...... 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 of sensitivity...... and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis....

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

DEFF Research Database (Denmark)

Sultan, Abida; Andersen, Birgit; Svensson, Birte

2016-01-01

Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant...... xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC–MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface......-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation...

The use of activity based protein profiling to study proteasome biology

NARCIS (Netherlands)

Paniagua Soriano, Guillem

2016-01-01

The work described in this thesis focuses on the characterization of proteasome directed activity-based probes (ABPs) as well as on the adaptation mechanisms that make multiple myeloma derived cell lines resistant against proteasome inhibitors (PIs).

Cellular reprogramming through mitogen-activated protein kinases

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Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Profiling physical activity motivation based on self-determination theory: a cluster analysis approach.

Science.gov (United States)

Friederichs, Stijn Ah; Bolman, Catherine; Oenema, Anke; Lechner, Lilian

2015-01-01

In order to promote physical activity uptake and maintenance in individuals who do not comply with physical activity guidelines, it is important to increase our understanding of physical activity motivation among this group. The present study aimed to examine motivational profiles in a large sample of adults who do not comply with physical activity guidelines. The sample for this study consisted of 2473 individuals (31.4% male; age 44.6 ± 12.9). In order to generate motivational profiles based on motivational regulation, a cluster analysis was conducted. One-way analyses of variance were then used to compare the clusters in terms of demographics, physical activity level, motivation to be active and subjective experience while being active. Three motivational clusters were derived based on motivational regulation scores: a low motivation cluster, a controlled motivation cluster and an autonomous motivation cluster. These clusters differed significantly from each other with respect to physical activity behavior, motivation to be active and subjective experience while being active. Overall, the autonomous motivation cluster displayed more favorable characteristics compared to the other two clusters. The results of this study provide additional support for the importance of autonomous motivation in the context of physical activity behavior. The three derived clusters may be relevant in the context of physical activity interventions as individuals within the different clusters might benefit most from different intervention approaches. In addition, this study shows that cluster analysis is a useful method for differentiating between motivational profiles in large groups of individuals who do not comply with physical activity guidelines.

Multiplex single-molecule interaction profiling of DNA barcoded proteins

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

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

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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.

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

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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

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

Directory of Open Access Journals (Sweden)

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

Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions.

Science.gov (United States)

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

2013-12-03

Pyrethroid insecticides are used to control 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 nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or "pyrethrome." Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in 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 unique tools for disease control.

DTFP-Growth: Dynamic Threshold-Based FP-Growth Rule Mining Algorithm Through Integrating Gene Expression, Methylation, and Protein-Protein Interaction Profiles.

Science.gov (United States)

Mallik, Saurav; Bhadra, Tapas; Mukherji, Ayan; Mallik, Saurav; Bhadra, Tapas; Mukherji, Ayan; Mallik, Saurav; Bhadra, Tapas; Mukherji, Ayan

2018-04-01

Association rule mining is an important technique for identifying interesting relationships between gene pairs in a biological data set. Earlier methods basically work for a single biological data set, and, in maximum cases, a single minimum support cutoff can be applied globally, i.e., across all genesets/itemsets. To overcome this limitation, in this paper, we propose dynamic threshold-based FP-growth rule mining algorithm that integrates gene expression, methylation and protein-protein interaction profiles based on weighted shortest distance to find the novel associations among different pairs of genes in multi-view data sets. For this purpose, we introduce three new thresholds, namely, Distance-based Variable/Dynamic Supports (DVS), Distance-based Variable Confidences (DVC), and Distance-based Variable Lifts (DVL) for each rule by integrating co-expression, co-methylation, and protein-protein interactions existed in the multi-omics data set. We develop the proposed algorithm utilizing these three novel multiple threshold measures. In the proposed algorithm, the values of , , and are computed for each rule separately, and subsequently it is verified whether the support, confidence, and lift of each evolved rule are greater than or equal to the corresponding individual , , and values, respectively, or not. If all these three conditions for a rule are found to be true, the rule is treated as a resultant rule. One of the major advantages of the proposed method compared with other related state-of-the-art methods is that it considers both the quantitative and interactive significance among all pairwise genes belonging to each rule. Moreover, the proposed method generates fewer rules, takes less running time, and provides greater biological significance for the resultant top-ranking rules compared to previous methods.

Effects of Holder pasteurization on the protein profile of human milk.

Science.gov (United States)

Peila, Chiara; Coscia, Alessandra; Bertino, Enrico; Cavaletto, Maria; Spertino, Stefano; Icardi, Sara; Tortone, Claudia; Visser, Gerard H A; Gazzolo, Diego

2016-04-07

The most widespread method for the treatment of donor milk is the Holder pasteurization (HoP). The available literature data show that HoP may cause degradation of some bioactive components. The aim of this study was to determine the effect of HoP on the protein profile of human milk (HM) using a GeLC-MS method, a proteomic approach and a promising technique able to offer a qualitative HM protein profile. HM samples were collected by standardized methods from 20 mothers carrying both preterm and term newborns. A aliquot of each sample was immediately frozen at -80 °C, whilst another one was Holder pasteurized and then frozen. All samples were then analyzed by GeLC-MS. The protein bands of interest were excised from the gel, digested with trypsin and identified by nano-HPLC-MS/MS analysis. The protein profile before and after HoP showed qualitative differences only in 6 samples out of 20, while in the remaining 14 no detectable differences were found. The differences interested only colostrums and transitional milk samples and regarded the decrease of the electrophoretic bands corresponding to alpha and beta-casein, tenascin, lactoferrin and immunoglobulin. In the majority of samples, HoP did not cause any modification, thereby preserving the biological activity of HM proteins.

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Science.gov (United States)

Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

2012-04-06

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Protein-based nanostructures as carriers for photo-physically active molecules in biosystems

OpenAIRE

Delcanale, Pietro

2017-01-01

In nature, many proteins function as carriers, being able to bind, transport and possibly release a ligand within a biological system. Protein-based carriers are interesting systems for drug delivery, with the remarkable advantage of being water-soluble and, as inherent components of biosystems, highly bio-compatible. This work focuses on the use of protein-based carriers for the delivery of hydrophobic photo-physically active molecules, whose structure and chemical properties lead to spontan...

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

Institute of Scientific and Technical Information of China (English)

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.

Analysis of protein profiles using fuzzy clustering methods

DEFF Research Database (Denmark)

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

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...

Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

Energy Technology Data Exchange (ETDEWEB)

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.

Changes in the protein profile of Habanero pepper ( Capsicum ...

African Journals Online (AJOL)

Protein profile was studied during the development of Capsicum chinense somatic embryos. The total protein content and profile of polypeptides (by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of somatic embryos at different developmental stages (globular, heart-shaped, torpedo and cotyledonary stages) ...

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

Energy Technology Data Exchange (ETDEWEB)

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.

Computational Identification of Protein Pupylation Sites by Using Profile-Based Composition of k-Spaced Amino Acid Pairs.

Directory of Open Access Journals (Sweden)

Md Mehedi Hasan

Full Text Available Prokaryotic proteins are regulated by pupylation, a type of post-translational modification that contributes to cellular function in bacterial organisms. In pupylation process, the prokaryotic ubiquitin-like protein (Pup tagging is functionally analogous to ubiquitination in order to tag target proteins for proteasomal degradation. To date, several experimental methods have been developed to identify pupylated proteins and their pupylation sites, but these experimental methods are generally laborious and costly. Therefore, computational methods that can accurately predict potential pupylation sites based on protein sequence information are highly desirable. In this paper, a novel predictor termed as pbPUP has been developed for accurate prediction of pupylation sites. In particular, a sophisticated sequence encoding scheme [i.e. the profile-based composition of k-spaced amino acid pairs (pbCKSAAP] is used to represent the sequence patterns and evolutionary information of the sequence fragments surrounding pupylation sites. Then, a Support Vector Machine (SVM classifier is trained using the pbCKSAAP encoding scheme. The final pbPUP predictor achieves an AUC value of 0.849 in 10-fold cross-validation tests and outperforms other existing predictors on a comprehensive independent test dataset. The proposed method is anticipated to be a helpful computational resource for the prediction of pupylation sites. The web server and curated datasets in this study are freely available at http://protein.cau.edu.cn/pbPUP/.

Profile of Students' Creative Thinking Skills on Quantitative Project-Based Protein Testing using Local Materials

Directory of Open Access Journals (Sweden)

D. K. Sari

2017-04-01

Full Text Available The purpose of this study is to obtain a profile of students’ creative thinking skills on quantitative project-based protein testing using local materials. Implementation of the research is using quasi-experimental method pre-test post-test control group design with 40 students involved in Biochemistry lab. The research instrument is pre-test and post-test using creative thinking skills in the form of description and students’ questionnaire. The analysis was performed with SPSS 22.0 program to see the significance normality, U Mann-Whitney test for nonparametric statistics, N-Gain score, and the percentage of student responses to the practicum performed. The research result shows that the pretest rate in the experimental group is 8.25 while in the control group is 6.90. After attending a project-based practicum with local materials, the experimental group obtained the mean of posttest is 37.55 while in control class is 11.18. The students’ improvement on creative thinking skills can be seen from the average of N-Gain in the experimental class with 0.32 (medium category and in the control category with 0.05 (low category. The experimental and control class have different creative thinking skills significantly different fluency, flexibility, novelty, and detail. It can be concluded that quantitative project-based protein testing using local materials can improve students’ creative thinking skills. 71% of total students feel that quantitative project-based protein testing using local materials make them more creative in doing a practicum in the laboratory.

Profile-IQ: Web-based data query system for local health department infrastructure and activities.

Science.gov (United States)

Shah, Gulzar H; Leep, Carolyn J; Alexander, Dayna

2014-01-01

To demonstrate the use of National Association of County & City Health Officials' Profile-IQ, a Web-based data query system, and how policy makers, researchers, the general public, and public health professionals can use the system to generate descriptive statistics on local health departments. This article is a descriptive account of an important health informatics tool based on information from the project charter for Profile-IQ and the authors' experience and knowledge in design and use of this query system. Profile-IQ is a Web-based data query system that is based on open-source software: MySQL 5.5, Google Web Toolkit 2.2.0, Apache Commons Math library, Google Chart API, and Tomcat 6.0 Web server deployed on an Amazon EC2 server. It supports dynamic queries of National Profile of Local Health Departments data on local health department finances, workforce, and activities. Profile-IQ's customizable queries provide a variety of statistics not available in published reports and support the growing information needs of users who do not wish to work directly with data files for lack of staff skills or time, or to avoid a data use agreement. Profile-IQ also meets the growing demand of public health practitioners and policy makers for data to support quality improvement, community health assessment, and other processes associated with voluntary public health accreditation. It represents a step forward in the recent health informatics movement of data liberation and use of open source information technology solutions to promote public health.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Science.gov (United States)

Jameson, Eleanor; Taubert, Martin; Coyotzi, Sara; Chen, Yin; Eyice, Özge; Schäfer, Hendrik; Murrell, J Colin; Neufeld, Josh D; Dumont, Marc G

2017-01-01

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13 C, 18 O, or 15 N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies

Protein expression profiling of inflammatory mediators in human temporal lobe epilepsy reveals co-activation of multiple chemokines and cytokines

Directory of Open Access Journals (Sweden)

Kan Anne A

2012-08-01

Full Text Available Abstract Mesial temporal lobe epilepsy (mTLE is a chronic and often treatment-refractory brain disorder characterized by recurrent seizures originating from the hippocampus. The pathogenic mechanisms underlying mTLE remain largely unknown. Recent clinical and experimental evidence supports a role of various inflammatory mediators in mTLE. Here, we performed protein expression profiling of 40 inflammatory mediators in surgical resection material from mTLE patients with and without hippocampal sclerosis, and autopsy controls using a multiplex bead-based immunoassay. In mTLE patients we identified 21 upregulated inflammatory mediators, including 10 cytokines and 7 chemokines. Many of these upregulated mediators have not previously been implicated in mTLE (for example, CCL22, IL-7 and IL-25. Comparing the three patient groups, two main hippocampal expression patterns could be distinguished, pattern I (for example, IL-10 and IL-25 showing increased expression in mTLE + HS patients compared to mTLE-HS and controls, and pattern II (for example, CCL4 and IL-7 showing increased expression in both mTLE groups compared to controls. Upregulation of a subset of inflammatory mediators (for example, IL-25 and IL-7 could not only be detected in the hippocampus of mTLE patients, but also in the neocortex. Principle component analysis was used to cluster the inflammatory mediators into several components. Follow-up analyses of the identified components revealed that the three patient groups could be discriminated based on their unique expression profiles. Immunocytochemistry showed that IL-25 IR (pattern I and CCL4 IR (pattern II were localized in astrocytes and microglia, whereas IL-25 IR was also detected in neurons. Our data shows co-activation of multiple inflammatory mediators in hippocampus and neocortex of mTLE patients, indicating activation of multiple pro- and anti-epileptogenic immune pathways in this disease.

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-05-01

Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

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

DEFF Research Database (Denmark)

Juncker, Agnieszka; Larsen, Mette Voldby; Weinhold, Nils

2009-01-01

Background: Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable. Methodology/Principal Finding: In the present large......-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...

Development of antimicrobial active packaging materials based on gluten proteins.

Science.gov (United States)

Gómez-Heincke, Diana; Martínez, Inmaculada; Partal, Pedro; Guerrero, Antonio; Gallegos, Críspulo

2016-08-01

The incorporation of natural biocide agents into protein-based bioplastics, a source of biodegradable polymeric materials, manufactured by a thermo-mechanical method is a way to contribute to a sustainable food packaging industry. This study assesses the antimicrobial activity of 10 different biocides incorporated into wheat gluten-based bioplastics. The effect that formulation, processing, and further thermal treatments exert on the thermo-mechanical properties, water absorption characteristics and rheological behaviour of these materials is also studied. Bioplastics containing six of the 10 examined bioactive agents have demonstrated suitable antimicrobial activity at 37 °C after their incorporation into the bioplastic. Moreover, the essential oils are able to create an antimicrobial atmosphere within a Petri dish. Depending on the selected biocide, its addition may alter the bioplastics protein network in a different extent, which leads to materials exhibiting less water uptake and different rheological and thermo-mechanical behaviours. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Analysis of protein profiles in diabetic rat blood plasma that induced by alloxan

Science.gov (United States)

Hidayati, Dewi; Abdulgani, Nurlita; Setiyawan, Hengki; Trisnawati, Indah; Ashuri, Nova Maulidina; Sa'adah, Noor Nailis

2017-06-01

Proteomics is the study to identify the proteins involved in physiological metabolic pathway. The protein profiles of blood plasma from alloxan-induced diabetic rats has investigated using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Data were analyzed descriptively based on variations of the type and intensity of the protein. There were identified the similarity of protein variant between diabetic and control rats included ankyrin (200kDa), IgG (150kDa), nephrin (136 kDa), IDE (112 kDA), albumin (66 kDa), prealbumin (55 kDA), CICP (43 kDa), ApoA-V (39 kDa), GAPDH (35 kDa), C-RP (27,1 kDa), leptin (16 kDa) and apelin (13 kDa). However, the apelin profile at diabetic rats shows the higher intensity than control.

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

Science.gov (United States)

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

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

Science.gov (United States)

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

2016-06-20

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/.

Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe

Directory of Open Access Journals (Sweden)

Ying Chen

2017-08-01

Full Text Available Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress. Keywords: Lipid-derived electrophile, 4-hydroxy-2-nonenal, Chemoproteomics, Aminooxy probe, Activity-based protein profiling

Insights into the immune manipulation mechanisms of pollen allergens by protein domain profiling.

Science.gov (United States)

Patel, Seema; Rani, Aruna; Goyal, Arun

2017-10-01

Plant pollens are airborne allergens, as their inhalation causes immune activation, leading to rhinitis, conjunctivitis, sinusitis and oral allergy syndrome. A myriad of pollen proteins belonging to profilin, expansin, polygalacturonase, glucan endoglucosidase, pectin esterase, and lipid transfer protein class have been identified. In the present in silico study, the protein domains of fifteen pollen sequences were extracted from the UniProt database and submitted to the interactive web tool SMART (Simple Modular Architecture Research Tool), for finding the protein domain profiles. Analysis of the data based on custom-made scripts revealed the conservation of pathogenic domains such as OmpH, PROF, PreSET, Bet_v_1, Cpl-7 and GAS2. Further, the retention of critical domains like CHASE2, Galanin, Dak2, DALR_1, HAMP, PWI, EFh, Excalibur, CT, PbH1, HELICc, and Kelch in pollen proteins, much like cockroach allergens and lethal viruses (such as HIV, HCV, Ebola, Dengue and Zika) was observed. Based on the shared motifs in proteins of taxonomicall-ydispersed organisms, it can be hypothesized that allergens and pathogens manipulate the human immune system in a similar manner. Allergens, being inanimate, cannot replicate in human body, and are neutralized by immune system. But, when the allergens are unremitting, the immune system becomes persistently hyper-sensitized, creating an inflammatory milieu. This study is expected to contribute to the understanding of pollen allergenicity and pathogenicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Patterns of screen-based sedentary behavior and physical activity and associations with overweight among Norwegian adolescents: a latent profile approach

Directory of Open Access Journals (Sweden)

Ole Melkevik

2011-03-01

Full Text Available Background: Physical activity and screen based sedentary behaviors are both related to energy balance and to risk for becoming overweight. The aim of this study is to find out if these behaviors cluster together in order to find out whether groups of adolescents have particularly unfortunate levels of both physical activity and screen-based sedentary behaviors. Methods: Data are from the Norwegian 2005/2006 sample of the international "Health Behaviour in School-aged Children (HBSC study; A WHO cross-National Survey". Data were collected through questionnaires from 13-, 15- and 16-year-olds. The final sample included 4848 adolescents. Gender-stratified latent profile analysis was used to identify the different profiles. Results: Six profiles were identified for both boys and girls. Less than 30% of adolescents were found to have behavioral patterns which were associated with higher risk for overweight relative to the most healthy behavioral profile. Physical activity and screen-based sedentary behaviors cluster together in different ways suggesting independence between the behaviors. Low levels of physical activity was the most important predictor for overweight among boys. Screen-based sedentary behaviors were more important predictors of overweight among girls. Conclusions: Physical activity and screen-based sedentary behaviors are independent behaviors and may cluster together in manners which lead to low energy expenditure and subsequent increased risk for overweight among adolescents.

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

International Nuclear Information System (INIS)

Higa, A.M.; Noronha, M.D.N.; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L.

2008-01-01

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 ∼ 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

Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

Directory of Open Access Journals (Sweden)

Ines eLassowskat

2014-10-01

Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the PEN pathway required for penetration resistance to filamentous pathogens. Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org.

Petunia nectar proteins have ribonuclease activity.

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Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

2010-06-01

Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar.

Genetic characteristic of swamp buffalo (Bubalus bubalis) from Pampangan, South Sumatra based on blood protein profile

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Windusari, Yuanita; Hanum, Laila; Wahyudi, Rizki

2017-11-01

Swamp buffalo (Bubalus bubalis) is an endemic species and one of the genetic wealth of South Sumatra with a distribution area in the district of Pampangan (OganIlir and OganOganIlir). Suspected inbreeding causes decreased phenotypic properties. Inbreeding among various swamp buffalo is certainly not only lower the qualities but also genotypes and phenotypes. It is of interest to determine kinship variants swamp buffaloes from Pampangan through the analysis of a blood protein profile. Blood protein profile of four variants swamps buffalo was studied by using five electrophoresis system i.e. pre-albumin (Palb), albumin (Alb), ceruloplasmin (Cp), transferrin (Tf) and transferrin post (Ptf). In this paper, it is obtained that there was no significant differences among the four variants of the buffaloes were used as a sample. Prealbumin has two alleles (Palb1 and Palb2), albumin has three alleles (Alba, AlbB, AlbC), ceruloplasmin has one allele (BPA), post-transferrin has one allele (PTFA) with an allele frequency 1.0000 at any time transferrin has two alleles (TFA and TFB) with the allele frequency of 0.7500 and 1.0000. Characteristics prealbumin (Palb), albumin (Alb), ceruloplasmin (Cp), and post-transferrin (P-tf) is monomorphic, while transferrin is polymorphic average heterozygosity values all loci (H) 0.1286. Based on average heterozygosity, the swamp buffalo (Bubalusbubalis) from Pampangan has low genetic variation and closest genetic relationship.

Atmospheric profiles from active space-based radio measurements

Science.gov (United States)

Hardy, Kenneth R.; Hinson, David P.; Tyler, G. L.; Kursinski, E. R.

1992-01-01

The paper describes determinations of atmospheric profiles from space-based radio measurements and the retrieval methodology used, with special attention given to the measurement procedure and the characteristics of the soundings. It is speculated that reliable profiles of the terrestrial atmosphere can be obtained by the occultation technique from the surface to a height of about 60 km. With the full complement of 21 the Global Positioning System (GPS) satellites and one GPS receiver in sun synchronous polar orbit, a maximum of 42 soundings could be obtained for each complete orbit or about 670 per day, providing almost uniform global coverage.

Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations.

Science.gov (United States)

Dissanayake, Thakshila; Swails, Jason M; Harris, Michael E; Roitberg, Adrian E; York, Darrin M

2015-02-17

The measurement of reaction rate as a function of pH provides essential information about mechanism. These rates are sensitive to the pK(a) values of amino acids directly involved in catalysis that are often shifted by the enzyme active site environment. Experimentally observed pH-rate profiles are usually interpreted using simple kinetic models that allow estimation of "apparent pK(a)" values of presumed general acid and base catalysts. One of the underlying assumptions in these models is that the protonation states are uncorrelated. In this work, we introduce the use of constant pH molecular dynamics simulations in explicit solvent (CpHMD) with replica exchange in the pH-dimension (pH-REMD) as a tool to aid in the interpretation of pH-activity data of enzymes and to test the validity of different kinetic models. We apply the methods to RNase A, a prototype acid-base catalyst, to predict the macroscopic and microscopic pK(a) values, as well as the shape of the pH-rate profile. Results for apo and cCMP-bound RNase A agree well with available experimental data and suggest that deprotonation of the general acid and protonation of the general base are not strongly coupled in transphosphorylation and hydrolysis steps. Stronger coupling, however, is predicted for the Lys41 and His119 protonation states in apo RNase A, leading to the requirement for a microscopic kinetic model. This type of analysis may be important for other catalytic systems where the active forms of the implicated general acid and base are oppositely charged and more highly correlated. These results suggest a new way for CpHMD/pH-REMD simulations to bridge the gap with experiments to provide a molecular-level interpretation of pH-activity data in studies of enzyme mechanisms.

Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

Science.gov (United States)

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-01

Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

Differences of protein profile before and after orthodontic treatment

Science.gov (United States)

Nasri, Farah Amirah Mohd; Wahab, Rohaya Megat Abdul; Karsani, Saiful Anuar; Ariffin, Shahrul Hisham Zainal

2016-11-01

Mechanical forces in orthodontic treatment used to treat malocclusion can cause inflamed gingival tissue and the process of tooth movement may resorb dental root. Root resorption is an iatrogenic effect of orthodontic treatment but it can be monitored using protein biomarker. This study aims to investigate the differences of protein profile before and after orthodontic treatment using different staining methods. Human gingival crevicular fluid and saliva were collected from orthodontic patients before and after treatment. Protein profile were observed using SDS-PAGE. Our study shows down regulation of proteins after 3 months of treatment. Hence, there are potential values from this study to aid in investigation for specific biomarkers for root resorption.

Genotypic variability and mutant identification in cicer arietinum L. by seed storage protein profiling

International Nuclear Information System (INIS)

Hameed, A.; Iqbal, N.; Shah, T.M.

2012-01-01

A collection of thirty-four chickpea genotypes, including five kabuli and twenty-nine desi, were analyzed by SDS-PAGE for seed storage protein profiling. Total soluble seed proteins were resolved on 12% gels. A low level of variability was observed in desi as compared to kabuli genotypes. Dendrogram based on electrophoretic data clustered the thirty-four genotypes in four major groups. As large number of desi genotypes illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. One kabuli genotype ILC-195 found to be the most divergent showing 86% similarity with all other genotypes. ILC-195 can be distinguished from its mutant i.e., CM-2000 and other kabuli genotypes on the basis of three peptides i.e. SSP-66, SSP-43 and SSP-39. Some proteins peptides were found to be genotype specific like SSP-26 for ICCV-92311. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in chickpea. Among 33 observed peptides, only six seed storages proteins from chickpea source were available in databases. On the basis of molecular weight similarity, identified peptides were SSP-64 as Serine/Threonine dehydratase, SSP-56 as Alpha-amylase inhibitor, SSP-50 as Provicillin, SSP-39 as seed imbibition protein, SSP-35 as Isoflavane reductase and SSP-19 as lipid transport protein. Highest variability was observed in vicillin subunits and beta subunits of legumins and its polymorphic forms. In conclusion, seed storage profiling can be economically used to asses the genetic variation, phylogenetic relationship and as markers to differentiate mutants from their parents. (author)

Defatted Detarium senegalense seed-based diet alters lipid profile ...

African Journals Online (AJOL)

Defatted Detarium senegalense seed-based diet alters lipid profile, ... cheaper alternative source for good quality protein for dietary purposes, we evaluated Detarium ... Whole seed residue, DDS seed flour and control diets (soybeans) were ...

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

Science.gov (United States)

da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

2017-08-09

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Prioritization of candidate disease genes by topological similarity between disease and protein diffusion profiles.

Science.gov (United States)

Zhu, Jie; Qin, Yufang; Liu, Taigang; Wang, Jun; Zheng, Xiaoqi

2013-01-01

Identification of gene-phenotype relationships is a fundamental challenge in human health clinic. Based on the observation that genes causing the same or similar phenotypes tend to correlate with each other in the protein-protein interaction network, a lot of network-based approaches were proposed based on different underlying models. A recent comparative study showed that diffusion-based methods achieve the state-of-the-art predictive performance. In this paper, a new diffusion-based method was proposed to prioritize candidate disease genes. Diffusion profile of a disease was defined as the stationary distribution of candidate genes given a random walk with restart where similarities between phenotypes are incorporated. Then, candidate disease genes are prioritized by comparing their diffusion profiles with that of the disease. Finally, the effectiveness of our method was demonstrated through the leave-one-out cross-validation against control genes from artificial linkage intervals and randomly chosen genes. Comparative study showed that our method achieves improved performance compared to some classical diffusion-based methods. To further illustrate our method, we used our algorithm to predict new causing genes of 16 multifactorial diseases including Prostate cancer and Alzheimer's disease, and the top predictions were in good consistent with literature reports. Our study indicates that integration of multiple information sources, especially the phenotype similarity profile data, and introduction of global similarity measure between disease and gene diffusion profiles are helpful for prioritizing candidate disease genes. Programs and data are available upon request.

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

Science.gov (United States)

Souda, Puneet; Ryan, Christopher M.; Cramer, William A.; Whitelegge, Julian

2011-01-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein’s native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. PMID:21982782

Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

Science.gov (United States)

Wan, Cen; Lees, Jonathan G; Minneci, Federico; Orengo, Christine A; Jones, David T

2017-10-01

Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Cen Wan

2017-10-01

Full Text Available Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

O-GlcNAc profiling: from proteins to proteomes

Science.gov (United States)

2014-01-01

O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

A new impedance based approach to test the activity of recombinant protein--Semaphorins as a test case.

Science.gov (United States)

Birger, Anastasya; Besser, Elazar; Reubinoff, Benjamin; Behar, Oded

2015-10-01

The biological activity of a recombinant protein is routinely measured using a bioassay such as an enzyme assay. However, many proteins have no enzymatic activity and in many cases it is difficult to devise a simple and reliable approach to test their activity. Semaphorins, Ephrins, Slits, Netrins or amylin-assisted proteins have numerous activities affecting many systems and cell types in the human body. Most of them are also able to induce rapid cytoskeleton changes at least in some cell types. We assumed therefore, that such proteins might be tested based on their ability to modulate the cytoskeleton. Here we tested a number of semaphorins in an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes. This system has proved to be a very fast, sensitive and effective way to monitor and determine the activity of such proteins. Furthermore we showed that it is possible to customize a cell-protein system by transfecting the cells with specific receptors and test the cell response following the addition of the recombinant ligand protein. Since other protein families such as Ephrins and Netrins can also influence the cytoskeleton of some cells, this approach may be applicable to a large number of proteins. Copyright © 2015 Elsevier GmbH. All rights reserved.

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

KAUST Repository

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.

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

Science.gov (United States)

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

2014-11-27

In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

Science.gov (United States)

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

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

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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.

Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

DEFF Research Database (Denmark)

Callesen, Anne K; Mohammed, Shabaz; Bunkenborg, Jakob

2005-01-01

for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI...

Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

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Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

2011-01-01

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

Oak protein profile alterations upon root colonization by an ectomycorrhizal fungus

DEFF Research Database (Denmark)

Sebastiana, Mónica; Martins, Joana; Figueiredo, Andreia

2017-01-01

in the roots. Consistent with the results of the biochemical analysis, the proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose for the metabolic activity of mycorrhizal roots which is consistent with an increased allocation of carbohydrates from the plant to the fungus...... to ectomycorrhizae formation using a proteomics approach complemented by biochemical analysis of carbohydrate levels. Comparative proteome analysis between mycorrhizal and nonmycorrhizal cork oak plants revealed no differences at the foliar level. However, the protein profile of 34 unique oak proteins was altered...... in order to sustain the symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of defense reactions, increased nutrient mobilization from the plant-fungus interface (N and P), as well as cytoskeleton rearrangements and induction of plant cell wall loosening for fungal root...

ReTrOS: a MATLAB toolbox for reconstructing transcriptional activity from gene and protein expression data.

Science.gov (United States)

Minas, Giorgos; Momiji, Hiroshi; Jenkins, Dafyd J; Costa, Maria J; Rand, David A; Finkenstädt, Bärbel

2017-06-26

Given the development of high-throughput experimental techniques, an increasing number of whole genome transcription profiling time series data sets, with good temporal resolution, are becoming available to researchers. The ReTrOS toolbox (Reconstructing Transcription Open Software) provides MATLAB-based implementations of two related methods, namely ReTrOS-Smooth and ReTrOS-Switch, for reconstructing the temporal transcriptional activity profile of a gene from given mRNA expression time series or protein reporter time series. The methods are based on fitting a differential equation model incorporating the processes of transcription, translation and degradation. The toolbox provides a framework for model fitting along with statistical analyses of the model with a graphical interface and model visualisation. We highlight several applications of the toolbox, including the reconstruction of the temporal cascade of transcriptional activity inferred from mRNA expression data and protein reporter data in the core circadian clock in Arabidopsis thaliana, and how such reconstructed transcription profiles can be used to study the effects of different cell lines and conditions. The ReTrOS toolbox allows users to analyse gene and/or protein expression time series where, with appropriate formulation of prior information about a minimum of kinetic parameters, in particular rates of degradation, users are able to infer timings of changes in transcriptional activity. Data from any organism and obtained from a range of technologies can be used as input due to the flexible and generic nature of the model and implementation. The output from this software provides a useful analysis of time series data and can be incorporated into further modelling approaches or in hypothesis generation.

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

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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

Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

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Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

2013-05-13

Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against

Profiling the biological activity of oxide nanomaterials with mechanistic models

NARCIS (Netherlands)

Burello, E.

2013-01-01

In this study we present three mechanistic models for profiling the potential biological and toxicological effects of oxide nanomaterials. The models attempt to describe the reactivity, protein adsorption and membrane adhesion processes of a large range of oxide materials and are based on properties

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

Science.gov (United States)

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

Statistical potential-based amino acid similarity matrices for aligning distantly related protein sequences.

Science.gov (United States)

Tan, Yen Hock; Huang, He; Kihara, Daisuke

2006-08-15

Aligning distantly related protein sequences is a long-standing problem in bioinformatics, and a key for successful protein structure prediction. Its importance is increasing recently in the context of structural genomics projects because more and more experimentally solved structures are available as templates for protein structure modeling. Toward this end, recent structure prediction methods employ profile-profile alignments, and various ways of aligning two profiles have been developed. More fundamentally, a better amino acid similarity matrix can improve a profile itself; thereby resulting in more accurate profile-profile alignments. Here we have developed novel amino acid similarity matrices from knowledge-based amino acid contact potentials. Contact potentials are used because the contact propensity to the other amino acids would be one of the most conserved features of each position of a protein structure. The derived amino acid similarity matrices are tested on benchmark alignments at three different levels, namely, the family, the superfamily, and the fold level. Compared to BLOSUM45 and the other existing matrices, the contact potential-based matrices perform comparably in the family level alignments, but clearly outperform in the fold level alignments. The contact potential-based matrices perform even better when suboptimal alignments are considered. Comparing the matrices themselves with each other revealed that the contact potential-based matrices are very different from BLOSUM45 and the other matrices, indicating that they are located in a different basin in the amino acid similarity matrix space.

Angiotensin I-Converting Enzyme (ACE Inhibitory Activity, Antioxidant Properties, Phenolic Content and Amino Acid Profiles of Fucus spiralis L. Protein Hydrolysate Fractions

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Lisete Paiva

2017-10-01

Full Text Available Food protein-derived hydrolysates with multi-bioactivities such as antihypertensive and antioxidant properties have recently received special attention since both activities can play significant roles in preventing cardiovascular diseases. This study reports, for the first time, the angiotensin I-converting enzyme (ACE-inhibition and antioxidant properties of ultrafiltrate fractions (UF with different molecular weight ranges (<1, 1–3 and ≥3 kDa obtained from Fucus spiralis protein hydrolysate (FSPH digested with cellulase–bromelain. The amino acids profile, recovery yield, protein, peptide and total phenolic contents of these FSPH-UF, and the in vitro digestibility of F. spiralis crude protein were also investigated. FSPH-UF ≥3 kDa presented remarkably higher ACE-inhibition, yield, peptide and polyphenolic (phlorotannins contents. Antioxidant analysis showed that FSPH-UF <1 kDa and ≥3 kDa exhibited significantly higher scavenging of 2,2-diphenyl-1-picrylhydrazyl radical and ferrous ion-chelating (FIC activity. FSPH-UF ≥3 kDa had also notably higher ferric reducing antioxidant power (FRAP. Strong correlations were observed between ACE-inhibition and antioxidant activities (FIC and FRAP. The results suggest that ACE-inhibition and antioxidant properties of FSPH-UF may be due to the bioactive peptides and polyphenols released during the enzymatic hydrolysis. In conclusion, this study shows the potential use of defined size FSPH-UF for the prevention/treatment of hypertension and/or oxidative stress-related diseases.

SVM-PB-Pred: SVM based protein block prediction method using sequence profiles and secondary structures.

Science.gov (United States)

Suresh, V; Parthasarathy, S

2014-01-01

We developed a support vector machine based web server called SVM-PB-Pred, to predict the Protein Block for any given amino acid sequence. The input features of SVM-PB-Pred include i) sequence profiles (PSSM) and ii) actual secondary structures (SS) from DSSP method or predicted secondary structures from NPS@ and GOR4 methods. There were three combined input features PSSM+SS(DSSP), PSSM+SS(NPS@) and PSSM+SS(GOR4) used to test and train the SVM models. Similarly, four datasets RS90, DB433, LI1264 and SP1577 were used to develop the SVM models. These four SVM models developed were tested using three different benchmarking tests namely; (i) self consistency, (ii) seven fold cross validation test and (iii) independent case test. The maximum possible prediction accuracy of ~70% was observed in self consistency test for the SVM models of both LI1264 and SP1577 datasets, where PSSM+SS(DSSP) input features was used to test. The prediction accuracies were reduced to ~53% for PSSM+SS(NPS@) and ~43% for PSSM+SS(GOR4) in independent case test, for the SVM models of above two same datasets. Using our method, it is possible to predict the protein block letters for any query protein sequence with ~53% accuracy, when the SP1577 dataset and predicted secondary structure from NPS@ server were used. The SVM-PB-Pred server can be freely accessed through http://bioinfo.bdu.ac.in/~svmpbpred.

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

Science.gov (United States)

Sinclair, John; Timms, John F

2011-08-01

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

[The Hypo Ionic Protein Profile (HIPP). Laboratory analytical evaluation in Complementary and Alternative Medicine].

Science.gov (United States)

Berth, M; Stalpaert, M; Bosmans, E

2008-01-01

The hypo ionic protein profile (HIPP) is a test based on the reticulo-endothelial index of Sandor. We evaluated the analytical performance of this test by comparing the obtained data in the HIPP to the concentration of some frequently measured specific serum proteins. The alfa euglobulin zone mainly comprises of ceruloplasmin, complement factor 3, apolipoprotein B and haptoglobin. The beta and gamma euglobulin zone reflect the concentration of the immunoglobulins. Since these proteins cannot be distinguished from each other, the diagnostic value of the HIPP will be limited. The HIPP is an outdated and aspecific assay for protein measurements.

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

DEFF Research Database (Denmark)

Callesen, Anne; Madsen, Jonna S; Iachina, Maria

2010-01-01

In the present study, the use of a robust and sensitive mass spectrometry based protein profiling analysis was tested as diagnostic tools for women with an ovarian tumor. The potential additional diagnostic value of serum protein profiles independent of the information provided by CA125 were also...... investigated. Protein profiles of 113 serum samples from women with an ovarian tumor (54 malign and 59 benign) were generated using MALDI-TOF MS. A total of 98 peaks with a significant difference (pwomen with benign tumors/cysts and malignant ovarian tumors were identified. After...... average linkage clustering, a profile of 46 statistical significant mass peaks was identified to distinguish malignant tumors and benign tumors/cysts. In the subgroup of women with normal CA125 values (

Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.

Science.gov (United States)

Zhang, J; Wang, J; Lee, Y-M; Lim, T-K; Lin, Q; Shen, H-M

2017-01-01

Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy. © 2017 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

Cadd, Verity A; Hogg, Philip J; Harris, Adrian L; Feller, Stephan M

2006-01-01

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

3D profile-based approach to proteome-wide discovery of novel human chemokines.

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Aurelie Tomczak

Full Text Available Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides

Whole genome expression profiling associates activation of unfolded protein response with impaired production and release of epinephrine after recurrent hypoglycemia.

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Juhye Lena Kim

Full Text Available Recurrent hypoglycemia can occur as a major complication of insulin replacement therapy, limiting the long-term health benefits of intense glycemic control in type 1 and advanced type 2 diabetic patients. It impairs the normal counter-regulatory hormonal and behavioral responses to glucose deprivation, a phenomenon known as hypoglycemia associated autonomic failure (HAAF. The molecular mechanisms leading to defective counter-regulation are not completely understood. We hypothesized that both neuronal (excessive cholinergic signaling between the splanchnic nerve fibers and the adrenal medulla and humoral factors contribute to the impaired epinephrine production and release in HAAF. To gain further insight into the molecular mechanism(s mediating the blunted epinephrine responses following recurrent hypoglycemia, we utilized a global gene expression profiling approach. We characterized the transcriptomes during recurrent (defective counter-regulation model and acute hypoglycemia (normal counter-regulation group in the adrenal medulla of normal Sprague-Dawley rats. Based on comparison analysis of differentially expressed genes, a set of unique genes that are activated only at specific time points after recurrent hypoglycemia were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network indicated activation of the unfolded protein response. Furthermore, at least three additional pathways/interaction networks altered in the adrenal medulla following recurrent hypoglycemia were identified, which may contribute to the impaired epinephrine secretion in HAAF: greatly increased neuropeptide signaling (proenkephalin, neuropeptide Y, galanin; altered ion homeostasis (Na+, K+, Ca2+ and downregulation of genes involved in Ca2+-dependent exocytosis of secretory vesicles. Given the pleiotropic effects of the unfolded protein response in different organs, involved in maintaining glucose homeostasis, these

Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

International Nuclear Information System (INIS)

Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

2014-01-01

Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

The Skeletal Muscle Anabolic Response to Plant- versus Animal-Based Protein Consumption.

Science.gov (United States)

van Vliet, Stephan; Burd, Nicholas A; van Loon, Luc J C

2015-09-01

Clinical and consumer market interest is increasingly directed toward the use of plant-based proteins as dietary components aimed at preserving or increasing skeletal muscle mass. However, recent evidence suggests that the ingestion of the plant-based proteins in soy and wheat results in a lower muscle protein synthetic response when compared with several animal-based proteins. The possible lower anabolic properties of plant-based protein sources may be attributed to the lower digestibility of plant-based sources, in addition to greater splanchnic extraction and subsequent urea synthesis of plant protein-derived amino acids compared with animal-based proteins. The latter may be related to the relative lack of specific essential amino acids in plant- as opposed to animal-based proteins. Furthermore, most plant proteins have a relatively low leucine content, which may further reduce their anabolic properties when compared with animal proteins. However, few studies have actually assessed the postprandial muscle protein synthetic response to the ingestion of plant proteins, with soy and wheat protein being the primary sources studied. Despite the proposed lower anabolic properties of plant vs. animal proteins, various strategies may be applied to augment the anabolic properties of plant proteins. These may include the following: 1) fortification of plant-based protein sources with the amino acids methionine, lysine, and/or leucine; 2) selective breeding of plant sources to improve amino acid profiles; 3) consumption of greater amounts of plant-based protein sources; or 4) ingesting multiple protein sources to provide a more balanced amino acid profile. However, the efficacy of such dietary strategies on postprandial muscle protein synthesis remains to be studied. Future research comparing the anabolic properties of a variety of plant-based proteins should define the preferred protein sources to be used in nutritional interventions to support skeletal muscle mass gain

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

Science.gov (United States)

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

2015-03-03

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.

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

Energy Technology Data Exchange (ETDEWEB)

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.

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

DEFF Research Database (Denmark)

Callesen, Anne K; Christensen, René Depont; Madsen, Jonna S

2008-01-01

for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom......Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum......-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard...

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Science.gov (United States)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Cytotoxic activity of abietane diterpenoids from roots of Salvia sahendica by HPLC-based activity profiling

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Fahimeh Moradi-Afrapoli

Full Text Available ABSTRACT Screening of medicinal plants from Iranian flora against human cancer cell-lines have shown that an hexane extract from roots of Salvia sahendica Boiss. & Buhse, Lamiaceae, is active against human cervical cancer (HeLa and colorectal adenocarcinoma (Caco-2 cell-lines at the test concentration of 100 µg/ml (100% inhibition. Cytotoxicity of the extract was localized with the aid of HPLC-time-based activity profiling adapted to the tetrazolium colorimetric bioassay. Four abietane-type diterpenoids in active time-windows were identified as cytotoxic compounds namely: sahandone (1, sahandol (2, 12-deoxy-salvipisone (3 and sahandinone (4. Compound 1 showed the highest toxicity against HeLa cells (IC50 = 5.6 ± 0.1 µg/ml, which was comparable with betulinic acid (IC50 = 4.3 ± 1.2 µg/ml, the positive control. Compound 2 was active against the HeLa cells (IC50 = 8.9 ± 0.7 µg/ml but not the Caco-2 cell-line. Compounds 1, 3 and 4 exhibited moderate activity (IC50 = 22.9–41.4 µg/ml against the Caco-2 cells. This study reveals that the HeLa cells are more sensitive to all tested compounds than the Caco-2 cells. In silico molecular docking study showed a rigid binding of the compounds to tyrosine kinase pp60src, and proved their cytotoxic activity.

Pharmacology profiling of chemicals and proteins

DEFF Research Database (Denmark)

Kringelum, Jens Vindahl

between pharmaceuticals and proteins in vivo potential leads to unwanted adverse effects, toxicity and reduced half-life, but can also lead to novel therapeutic effects of already approved pharmaceuticals. Hence identification of in vivo targets is of importance in discovery, development and repurposing....... This limitation complicates adverse effect assessment in the early drug-development phase, thus contributing to drugattrition. Prediction models offer the possibility to close these gaps and provide more complete pharmacology profiles, however improvements in performances are required for these tools to serve...... to its nonself origin, which potentially alters the pharmacology profile of the substance. The neutralization of biopharmaceuticals by antidrug antibodies (ADAs) is an important element in the immune response cascade, however studies of ADA binding site on biopharmaceuticals, referred to as B...

New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

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Bartłomiej Dziuba

2014-08-01

Full Text Available New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM, random forest (RF, artificial neural networks (ANN and discriminant analysis (DA available in the Collection of Anti-Microbial Peptides (CAMP database. Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.

Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab.

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Kenta Mukaihara

Full Text Available Giant cell tumors of bone (GCTB are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the

Profil Protein Trypanosoma evansi dari Daerah Geografis Berbeda di Indonesia Tahun 2012-2014 dengan Sodium Dodecil Sulphate Polyacrylamide Gel Electrophoresis (TRYPANOSOMA EVANSI PROTEIN PROFILE OF DIFFERENT GEOGRAPHICAL AREAS ORIGIN IN INDONESIA

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Fitrine Ekawasti

2018-01-01

Full Text Available Surra outbreak in 2012 has led to more than 1,700 animals have died in the province of East Nusa Tenggara (NTT Indonesia. Surra case sporadically continues throughout the year in various areas, especially Kalimantan, Banten as well as other areas. Some reports reveal differences in protein profiles among multiple isolates of T. evansi. Therefore the purpose of this research were to find out the protein profile of each isolate T. evansi in Indonesia and the possible biological differences among them. Eleven isolates originating from the province of East Nusa Tenggara, South Kalimantan and Central Kalimantan, Banten, Lampung and Bengkulu has been isolated and purified Using DEAE. Trypanosoma isolate were frezeethawing repeatedly to obtain soluble protein. Furthermore, soluble protein is treated with heating or without heating and then each was run on SDS PAGE with Coomassie Blue staining. The protein profiles of all isolates were compared each other. The results showed that eleven isolates of T. evansi in Indonesia has a very diverse protein profile. Then for the purposes of development of diagnostic kit can be used whole lysate cell (WCL as stock antigen in serological test process.

The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids

OpenAIRE

Kreuzer, Johannes

2015-01-01

The target proteins of acivicin and structure derived probes in tumor cells were identified using activity-based protein profiling. The target proteins were further characterized and their relation to the antitumor activity of acivicin pointed out. In a further project, the activity of serine hydrolases in myoma and myometrium was examined from tissue samples. This revealed a different activity of mast cell proteases. Mittels Activity-based Protein Profiling wurde eine Identifikation der Z...

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Science.gov (United States)

González-García, Estefanía; Maly, Marek; de la Mata, Francisco Javier; Gómez, Rafael; Marina, María Luisa; García, María Concepción

2016-11-01

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.

Effects of Pineal Proteins on Biochemical, Enzyme Profile and Non ...

African Journals Online (AJOL)

Effects of Pineal Proteins on Biochemical, Enzyme Profile and Non-Specific Immune Response of Indian Goats under Thermal Stress. ... Total precipitated pineal proteins successfully and significantly relieved the animals from adverse effects of heat stress and metyrapone treatment. There is evidence that most of the ...

Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

Science.gov (United States)

Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

2015-06-01

Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

PROFIL PROTEIN HIPOFISA SAPI PERAH PERANAKAN FRIES HOLLAND (PFH BETINA FASE FOLIKULER DAN LUTEA

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Nurul Isnaini

2012-04-01

Full Text Available ABSTRAK   Penelitian dilakukan dengan tujuan untuk mengetahui profil protein hipofisa sapi perah PFH betina fase folikuler dan fase luteal dengan menggunakan metode SDS-PAGE. Hasil penelitian ini diharapkan dapat digunakan sebagai acuan untuk melaksanakan pengujian jenis protein tertentu yang terdapat dalam hipofisa sapi perah PFH.Materi yang digunakan dalam penelitian ini adalah hipofisa sapi perah PFH betina Fase Folikuler dan Fase Luteal. Sampel hipofisa didapatkan dari Rumah Potong Hewan (RPH Singosari Malang. Metode penelitian yang digunakan dalam penelitian ini adalah metode observasi. Dimana sampel hipofisa sapi perah PFH fase folikuler dan fase luteal dilakukan isolasi protein, dan ditentukan Berat Molekul (BM proteinnya. Data yang diperoleh dianalisis dengan menggunakan analisis diskriptif. Hasil penelitian menunjukkan bahwa profil protein hipofisa fase folikuler dan fase luteal sapi perah PFH memiliki perbedaan. Pada hipofisa fase folikuler memiliki 12 pita protein, yaitu 163; 112,3; 101,3; 85,8; 80,6; 71,2; 53,3; 45,2; 39,9; 32,4; 29,8; dan 24,8 kDa. Pada hipofisa fase luteal memiliki 12 pita protein, yaitu 163; 112,3; 101,3; 85,8; 71,2; 60,3; 53,3; 45,2; 39,9; 32,4; 29,8; dan 24,8 kDa. Pita protein yang membedakan adalah pita dengan berat molekul 80,6 kDa hanya terdapat pada hipofisa fase folikuler, dan pita dengan berat molekul 60,3 kDa hanya terdapat pada hipofisa fase luteal. Kesimpulan yang diperoleh dari penelitian ini adalah terdapat perbedaan profil protein yang dihasilkan pada hipofisa fase folikuler dengan hipofisa fase luteal. Saran yang diberikan adalah perlu dilakukan penelitian lanjutan yaitu uji immunoblothing atau westernblothing (WB untuk memastikan keberadaan  protein-protein tertentu.   Kata kunci: Hipofisa, folikuler, luteal, berat molekul protein. HIPOPHISIS PROTEIN PROFILE OF CROSSBREED FRIESH HOLLAND DAIRY CATTLE IN FOLLICULAR AND LUTEAL PHASE   ABSTRACT   The aim of this research was to identification of

Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

Science.gov (United States)

Hossler, Patrick; Racicot, Christopher; Chumsae, Christopher; McDermott, Sean; Cochran, Keith

2017-03-01

Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017. © 2017 American Institute of Chemical Engineers.

The association between the activity profile and cardiovascular risk.

Science.gov (United States)

Maddison, Ralph; Jiang, Yannan; Foley, Louise; Scragg, Robert; Direito, Artur; Olds, Timothy

2016-08-01

This study sought to better understand the interrelationships between physical activity and sedentary behaviour and the relationship to risk of cardiovascular disease (CVDR) in adults aged 30-75 years. Cross-sectional. Data from two-year waves (2003-2004 and 2005-2006) of the National Health and Nutritional Examination survey were analysed in 2014. Accelerometer-derived time and proportion of time spent sedentary and on moderate-to-vigorous physical activity (MVPA) were calculated to generate four activity profiles based on cut-points to define low and high levels for the respective behaviours. Using health outcome data, CVDR was calculated for each person. Weighted multiple linear regression models were used to evaluate the predicted effects of sedentary and physical activity behaviours on the CVDR score, adjusting for participants' sex, age group, race, annual household income, and accelerometer wear time. The lowest CVDR was observed among Busy Exercisers (high MVPA and low sedentary; 8.5%), whereas Couch Potatoes (low MVPA and high sedentary) had the highest (18.6%). Compared with the reference group (Busy Exercisers), the activity profile associated with the highest CVDR was Couch Potatoes (adjusted mean difference 3.6, SE 0.38, prisk landscape" was developed to better visualise the conjoint associations of MVPA and sedentary behaviour on CVDR for each activity profile. The association between MVPA was greater than that of sedentary behaviour; however, for people with low MVPA, shifts in sedentary behaviour may have the greatest impact on CVDR. Activity profiles that consider the interrelationships between physical activity and sedentary behaviour differ in terms of CVDR. Future interventions may need to be tailored to specific profiles and be dynamic enough to reflect change in the profile over time. Copyright © 2015 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the Western flower thrips, Frankliniella occidentalis.

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Zhi-Ke Zhang

Full Text Available Using RT-PCR and RACE-PCR strategies, we cloned and identified a new chemosensory protein (FoccCSP from the Western flower thrips, Frankliniella occidentalis, a species for which no chemosensory protein (CSP has yet been identified. The FoccCSP gene contains a 387 bp open-reading frame encoding a putative protein of 128 amino acids with a molecular weight of 14.51 kDa and an isoelectric point of 5.41. The deduced amino acid sequence contains a putative signal peptide of 19 amino acid residues at the N-terminus, as well as the typical four-cysteine signature found in other insect CSPs. As FoccCSP is from a different order of insect than other known CSPs, the GenBank FoccCSP homolog showed only 31-50% sequence identity with them. A neighbor-joining tree was constructed and revealed that FoccCSP is in a group with CSPs from Homopteran insects (e.g., AgosCSP4, AgosCSP10, ApisCSP, and NlugCSP9, suggesting that these genes likely developed from a common ancestral gene. The FoccCSP gene expression profile of different tissues and development stages was measured by quantitative real-time PCR. The results of this analysis revealed this gene is predominantly expressed in the antennae and also highly expressed in the first instar nymph, suggesting a function for FoccCSP in olfactory reception and in particular life activities during the first instar nymph stage. We expressed recombinant FoccCSP protein in a prokaryotic expression system and purified FoccCSP protein by affinity chromatography using a Ni-NTA-Sepharose column. Using N-phenyl-1-naphthylamine (1-NPN as a fluorescent probe in fluorescence-based competitive binding assay, we determined the binding affinities of 19 volatile substances for FoccCSP protein. This analysis revealed that anisic aldehyde, geraniol and methyl salicylate have high binding affinities for FoccCSP, with KD values of 10.50, 15.35 and 35.24 μM, respectively. Thus, our study indicates that FoccCSP may play an important role in

Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

Science.gov (United States)

Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

2009-04-17

Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

A ranking method for the concurrent learning of compounds with various activity profiles.

Science.gov (United States)

Dörr, Alexander; Rosenbaum, Lars; Zell, Andreas

2015-01-01

In this study, we present a SVM-based ranking algorithm for the concurrent learning of compounds with different activity profiles and their varying prioritization. To this end, a specific labeling of each compound was elaborated in order to infer virtual screening models against multiple targets. We compared the method with several state-of-the-art SVM classification techniques that are capable of inferring multi-target screening models on three chemical data sets (cytochrome P450s, dehydrogenases, and a trypsin-like protease data set) containing three different biological targets each. The experiments show that ranking-based algorithms show an increased performance for single- and multi-target virtual screening. Moreover, compounds that do not completely fulfill the desired activity profile are still ranked higher than decoys or compounds with an entirely undesired profile, compared to other multi-target SVM methods. SVM-based ranking methods constitute a valuable approach for virtual screening in multi-target drug design. The utilization of such methods is most helpful when dealing with compounds with various activity profiles and the finding of many ligands with an already perfectly matching activity profile is not to be expected.

Independent component analysis for the extraction of reliable protein signal profiles from MALDI-TOF mass spectra.

Science.gov (United States)

Mantini, Dante; Petrucci, Francesca; Del Boccio, Piero; Pieragostino, Damiana; Di Nicola, Marta; Lugaresi, Alessandra; Federici, Giorgio; Sacchetta, Paolo; Di Ilio, Carmine; Urbani, Andrea

2008-01-01

Independent component analysis (ICA) is a signal processing technique that can be utilized to recover independent signals from a set of their linear mixtures. We propose ICA for the analysis of signals obtained from large proteomics investigations such as clinical multi-subject studies based on MALDI-TOF MS profiling. The method is validated on simulated and experimental data for demonstrating its capability of correctly extracting protein profiles from MALDI-TOF mass spectra. The comparison on peak detection with an open-source and two commercial methods shows its superior reliability in reducing the false discovery rate of protein peak masses. Moreover, the integration of ICA and statistical tests for detecting the differences in peak intensities between experimental groups allows to identify protein peaks that could be indicators of a diseased state. This data-driven approach demonstrates to be a promising tool for biomarker-discovery studies based on MALDI-TOF MS technology. The MATLAB implementation of the method described in the article and both simulated and experimental data are freely available at http://www.unich.it/proteomica/bioinf/.

New insight into quinoa seed quality under salinity: changes in proteomic and amino acid profiles, phenolic content, and antioxidant activity of protein extracts

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Iris eAloisi

2016-05-01

Full Text Available Quinoa (Chenopodium quinoa Willd is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa seeds. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49 and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR, exposed to two levels of salinity (100 and 300 mM NaCl were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC and flavonoid (TFC contents, and antioxidant activity (AA of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace

New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts

Science.gov (United States)

Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B.; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

2016-01-01

Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the

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

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Ida Zahidah Irfan

2014-06-01

Full Text Available 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 by photometer principle using a commercial kit. There were significant (P<0.001 breed variation on total protein, albumin, globulin and albumin globulin ratio. Significant age differences were observed on total protein and albumin concentration (P<0.001, while globulin concentration and A/G ratio were also significant (P<0.05. Amongs groups of BCS, significant difference was verified only in the albumin concentration (P<0.05. The concentration of total proteins, albumins and globulins in the serum of the bulls are higher than standard values for cattle, while A/G ratio is lower.

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

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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.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-08-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. Four free amino acids were resolved using cation-exchange chromatography followed by detection with refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (uv) for tyrosine and phenylalanine. Amino acid detection by refractive index is not sensitive and uv absorbance detects only three amino acids. Derivatization of amino acids to make them detectable by uv absorbance enhances the applicability of OA/uv for the determination of enantiomeric ratios. The separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/uv is illustrated. Calculation of the specific rotation of 22 dansyl-L-amino acids shows that derivatization enhances the OA detectability of some amino acids but degrades that of others. RP-HPLC of proteins is a rapidly developing technique. Several researchers have reported the detection of multiple peaks when a pure protein is subjected to HPLC under certain conditions. These multiple peaks have been determined to be different conformations of the same protein. Since proteins are optically active, OA is a suitable detector. The RP-HPLC separation of conformers of soybean trypsin inhibitor is illustrated. Detection by OA/uv provides insights from the chromatogram unavailable from uv absorbance detection alone. In addition, identification of impurities is simplified with OA/uv. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation. 163 refs., 13 figs., 9 tabs.

Convolution based profile fitting

International Nuclear Information System (INIS)

Kern, A.; Coelho, A.A.; Cheary, R.W.

2002-01-01

Full text: In convolution based profile fitting, profiles are generated by convoluting functions together to form the observed profile shape. For a convolution of 'n' functions this process can be written as, Y(2θ)=F 1 (2θ)x F 2 (2θ)x... x F i (2θ)x....xF n (2θ). In powder diffractometry the functions F i (2θ) can be interpreted as the aberration functions of the diffractometer, but in general any combination of appropriate functions for F i (2θ) may be used in this context. Most direct convolution fitting methods are restricted to combinations of F i (2θ) that can be convoluted analytically (e.g. GSAS) such as Lorentzians, Gaussians, the hat (impulse) function and the exponential function. However, software such as TOPAS is now available that can accurately convolute and refine a wide variety of profile shapes numerically, including user defined profiles, without the need to convolute analytically. Some of the most important advantages of modern convolution based profile fitting are: 1) virtually any peak shape and angle dependence can normally be described using minimal profile parameters in laboratory and synchrotron X-ray data as well as in CW and TOF neutron data. This is possible because numerical convolution and numerical differentiation is used within the refinement procedure so that a wide range of functions can easily be incorporated into the convolution equation; 2) it can use physically based diffractometer models by convoluting the instrument aberration functions. This can be done for most laboratory based X-ray powder diffractometer configurations including conventional divergent beam instruments, parallel beam instruments, and diffractometers used for asymmetric diffraction. It can also accommodate various optical elements (e.g. multilayers and monochromators) and detector systems (e.g. point and position sensitive detectors) and has already been applied to neutron powder diffraction systems (e.g. ANSTO) as well as synchrotron based

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

Science.gov (United States)

Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

2009-12-24

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. 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. 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.

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

DEFF Research Database (Denmark)

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 ...

Reproducibility of mass spectrometry based protein profiles for diagnosis of ovarian cancer across clinical studies: A systematic review

DEFF Research Database (Denmark)

Callesen, AK; Mogensen, O; Jensen, AK

2012-01-01

of published discriminatory peaks to peaks found in an original MALDI MS protein profiling study was made to address the key question of reproducibility across studies. An overlap was found despite substantial heterogeneity between studies relating to study design, biological material, pre-analytical treatment...

Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

Science.gov (United States)

Kobayashi, Tetsuo; Yokoyama, Tomoko; Ito, Satoshi; Kobayashi, Daisuke; Yamagata, Akira; Okada, Moe; Oofusa, Ken; Narita, Ichiei; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

2014-11-01

Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P protein spots obtained, nine spots were significantly decreased in abundance at reassessment, corresponding to complement factor H, phospholipase D, serum amyloid A, complement component 4, and α-1-acid glycoprotein (P periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

Effect of soy protein on serum lipid profile and some lipid ...

African Journals Online (AJOL)

The effect of soy protein on serum lipid profile and some lipid metabolizing enzymes in rats fed with cholesterol diets was examined in this study. Rats were subjected to feeding trial over a period of six weeks on formulated diets containing: 20% soy protein with 0% cholesterol (group A), 20% soy protein with 5% cholesterol ...

Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

Science.gov (United States)

Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

2016-04-01

We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antimicrobial activity of biodegradable polysaccharide and protein-based films containing active agents.

Science.gov (United States)

Kuorwel, Kuorwel K; Cran, Marlene J; Sonneveld, Kees; Miltz, Joseph; Bigger, Stephen W

2011-04-01

Significant interest has emerged in the introduction of food packaging materials manufactured from biodegradable polymers that have the potential to reduce the environmental impacts associated with conventional packaging materials. Current technologies in active packaging enable effective antimicrobial (AM) packaging films to be prepared from biodegradable materials that have been modified and/or blended with different compatible materials and/or plasticisers. A wide range of AM films prepared from modified biodegradable materials have the potential to be used for packaging of various food products. This review examines biodegradable polymers derived from polysaccharides and protein-based materials for their potential use in packaging systems designed for the protection of food products from microbial contamination. A comprehensive table that systematically analyses and categorizes much of the current literature in this area is included in the review.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

Science.gov (United States)

2012-01-01

Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

Science.gov (United States)

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

In silico study of protein to protein interaction analysis of AMP-activated protein kinase and mitochondrial activity in three different farm animal species

Science.gov (United States)

Prastowo, S.; Widyas, N.

2018-03-01

AMP-activated protein kinase (AMPK) is cellular energy censor which works based on ATP and AMP concentration. This protein interacts with mitochondria in determine its activity to generate energy for cell metabolism purposes. For that, this paper aims to compare the protein to protein interaction of AMPK and mitochondrial activity genes in the metabolism of known animal farm (domesticated) that are cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In silico study was done using STRING V.10 as prominent protein interaction database, followed with biological function comparison in KEGG PATHWAY database. Set of genes (12 in total) were used as input analysis that are PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PPARGC1, ACC, CPT1B, NRF2 and SOD. The first 7 genes belong to gene in AMPK family, while the last 5 belong to mitochondrial activity genes. The protein interaction result shows 11, 8 and 5 metabolism pathways in Bos taurus, Sus scrofa and Gallus gallus, respectively. The top pathway in Bos taurus is AMPK signaling pathway (10 genes), Sus scrofa is Adipocytokine signaling pathway (8 genes) and Gallus gallus is FoxO signaling pathway (5 genes). Moreover, the common pathways found in those 3 species are Adipocytokine signaling pathway, Insulin signaling pathway and FoxO signaling pathway. Genes clustered in Adipocytokine and Insulin signaling pathway are PRKAA2, PPARGC1A, PRKAB1 and PRKAG2. While, in FoxO signaling pathway are PRKAA2, PRKAB1, PRKAG2. According to that, we found PRKAA2, PRKAB1 and PRKAG2 are the common genes. Based on the bioinformatics analysis, we can demonstrate that protein to protein interaction shows distinct different of metabolism in different species. However, further validation is needed to give a clear explanation.

Profiling and relationship of water-soluble sugar and protein compositions in soybean seeds.

Science.gov (United States)

Yu, Xiaomin; Yuan, Fengjie; Fu, Xujun; Zhu, Danhua

2016-04-01

Sugar and protein are important quality traits in soybean seeds for making soy-based food products. However, the investigations on both compositions and their relationship have rarely been reported. In this study, a total of 35 soybean germplasms collected from Zhejiang province of China, were evaluated for both water-soluble sugar and protein. The total water-soluble sugar (TWSS) content of the germplasms studied ranged from 84.70 to 140.91 mg/g and the water-soluble protein (WSP) content varied from 26.5% to 36.0%. The WSP content showed positive correlations with the TWSS and sucrose contents but negative correlations with the fructose and glucose contents. The clustering showed the 35 germplasms could be divided into four groups with specific contents of sugar and protein. The combination of water-soluble sugar and protein profiles provides useful information for future breeding and genetic research. This investigation will facilitate future work for seed quality improvement. Copyright © 2015. Published by Elsevier Ltd.

The activation mechanisms of G protein-coupled receptors : the case of the adenosine A2B and HCA2/3 receptors

NARCIS (Netherlands)

Liu, R.

2016-01-01

Identifying and elucidating the functions and activation of GPCRs will provide opportunities for novel drug discovery. We confirmed that a yeast system with an extended library of G proteins is very well suited for the study of GPCR activation, G protein coupling profiles, receptor-G protein binding

Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

DEFF Research Database (Denmark)

Callesen, Anne K; Madsen, Jonna S; Vach, Werner

2009-01-01

Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and ...

Detecting Immune System Response Proteins in a 500 Year-old Inca Mummy

Science.gov (United States)

Corthals, A.; Davalos, L.; Martin, D.W.; Rieger, R.; Chen, E.I.; Koller, A.

2011-01-01

Disease detection in ancient human samples currently relies on genomic-based assays, which are error prone due to contamination and cannot distinguish between active and latent pathogenic infection. On the other hand, protein-based assays such as global protein profiling offer complementary alternatives for the pathological diagnosis of archeological specimen. The discovery of three Inca mummies in 1998, perfectly preserved in the permafrost of the high Andes, allowed us to analyze mummy samples by protein-based and genomic-based assay. A buccal swab from one of the 500 year old mummy was analyzed by shotgun proteomics to detect the protein profile. Among the identified proteins, we found a signature of proteins indicating an immune response to a bacterial infection at the time of the mummy's death. Based on the external visible symptoms and the gamut of immune response proteins obtained from the mouth swab, we suspected that the pulmonary infection was caused by Mycobacterium. PCR assay followed by direct sequencing of the PCR products confirmed the presence of Mycobacterium sp. in the mouth swab. Until now, immunoassays have been the only way to detect an active immune response and infer infection in historical samples, but these were plagued by low specificity and sensitivity. However, we demonstrate here the feasibility of incorporating global protein profiling in the diagnosis of infection from archeological samples. Protein signatures obtained from these samples could be extremely useful in determining the status of infection while genomic-based assays can be used to detect the identity of the pathogen.

Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity

Directory of Open Access Journals (Sweden)

Zulezwan A. Malik

2013-12-01

Full Text Available Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC coupled to mass spectrometry (MS affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group bred as either high- or low-capacity runners (HCR and LCR, respectively that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001 in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897 and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5. Sixteen proteins were significantly (p < 0.05 more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH was 1

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

DEFF Research Database (Denmark)

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 estab...

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

Directory of Open Access Journals (Sweden)

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.

Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence.

Directory of Open Access Journals (Sweden)

Juliana Bernardes

2016-07-01

Full Text Available Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of

Venom Protein C activators as diagnostic agents for defects of protein C System.

Science.gov (United States)

Ramzan, Faiqah; Asmat, Andleeb

2018-06-18

Background Protein C is a vitamin K dependent plasma zymogen. It prevents clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converts the zymogen Protein C (PC) of vertebrates into activated PC, which has been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Conclusion The fast-acting PC activator Protac® from Agkistrodon contortrix (southern copperhead snake) venom has been found to have broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

Science.gov (United States)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

Continuous Water Vapor Profiles from Operational Ground-Based Active and Passive Remote Sensors

Science.gov (United States)

Turner, D. D.; Feltz, W. F.; Ferrare, R. A.

2000-01-01

The Atmospheric Radiation Measurement program's Southern Great Plains Cloud and Radiation Testbed site central facility near Lamont, Oklahoma, offers unique operational water vapor profiling capabilities, including active and passive remote sensors as well as traditional in situ radiosonde measurements. Remote sensing technologies include an automated Raman lidar and an automated Atmospheric Emitted Radiance Interferometer (AERI), which are able to retrieve water vapor profiles operationally through the lower troposphere throughout the diurnal cycle. Comparisons of these two water vapor remote sensing methods to each other and to radiosondes over an 8-month period are presented and discussed, highlighting the accuracy and limitations of each method. Additionally, the AERI is able to retrieve profiles of temperature while the Raman lidar is able to retrieve aerosol extinction profiles operationally. These data, coupled with hourly wind profiles from a 915-MHz wind profiler, provide complete specification of the state of the atmosphere in noncloudy skies. Several case studies illustrate the utility of these high temporal resolution measurements in the characterization of mesoscale features within a 3-day time period in which passage of a dryline, warm air advection, and cold front occurred.

ANALISIS PROFIL PROTEIN DARAH ANAK KAMBING PERANAKAN ETAWAH DENGAN PEMBERIAN PAKAN SUBSTITUSI SUSU SAPI

Directory of Open Access Journals (Sweden)

Teguh Wicaksono

2017-08-01

Full Text Available The objective of this study is to determine the protein profile of pre-weaning kids fed with cow's milk as a substitute for dam’s milk. The materials used were 18 Etawah Descendant (PE kids born the twin at the age of 5-13 days from 3-4-year-old dams. This experimental design was a completely randomized design with three treatments with six replications per treatment, namely the control (T0 fed 100% goat’s milk, treatment 1 (T1 fed 50% goat’s milk and 50% cow’s milk, treatment 2 (T2 fed 100% cow’s milk. The protein profile serum was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE method, 12,5% of the resolving gel and 3% of the stacking gel were used. The protein profile of the 5-14 days old PE kids were 19 protein bands with the molecular weight ranging from 15-160 kDa. The kids fed with 100% goat milk (T0 and those substituted by 50% cow's milk (T1, it was produced 19 protein bands with molecular weights ranging from 15 kDa to 155 kDa, while those fed with 100 % cow's milk (T2, it was produced 17 protein bands with molecular weights ranging from 13 kDa to 160 kDa. It can be concluded that the dam's milk substitute using cow's milk at the 50% level does not affect the blood protein profile of goat kids, while the 100% substitute produces the different number and types of protein

Protein profiles of hatchery egg shell membrane.

Science.gov (United States)

Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

2016-01-01

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

KAUST Repository

Ferreira, Ari J S

2014-06-12

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world\\'s oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

KAUST Repository

Ferreira, Ari J S; Siam, Rania; Setubal, Joã o C; Moustafa, Ahmed; Sayed, Ahmed; Chambergo, Felipe S; Dawe, Adam S; Ghazy, Mohamed A; Sharaf, Hazem; Ouf, Amged; Alam, Intikhab; Abdel-Haleem, Alyaa M; Lehvä slaiho, Heikki; Ramadan, Eman; Antunes, André ; Stingl, Ulrich; Archer, John A.C.; Jankovic, Boris R; Sogin, Mitchell; Bajic, Vladimir B.; El-Dorry, Hamza

2014-01-01

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

Directory of Open Access Journals (Sweden)

Ari J S Ferreira

Full Text Available Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

Comparative proteomics and protein profile related to phenolic compounds and antioxidant activity in germinated Oryza sativa 'KDML105' and Thai brown rice 'Mali Daeng' for better nutritional value.

Science.gov (United States)

Maksup, Sarunyaporn; Pongpakpian, Sarintip; Roytrakul, Sittiruk; Cha-Um, Suriyan; Supaibulwatana, Kanyaratt

2018-01-01

Brown rice (BR) and germinated brown rice (GBR) are considered as prime sources of carbohydrate and bioactive compounds for more than half of the populations worldwide. Several studies have reported on the proteomics of BR and GBR; however, the proteomic profiles related to the synthesis of bioactive compounds are less well documented. In the present study, BR and GBR were used in a comparative analysis of the proteomic and bioactive compound profiles for two famous Thai rice varieties: Khao Dawk Mali 105 (KDML) and Mali Daeng (MD). The proteomes of KDML and MD revealed differences in the expression patterns of proteins after germination. Total phenolic compound content, anthocyanin contents and antioxidant activity of red rice MD was approximately 2.6-, 2.2- and 9.2-fold higher, respectively, compared to that of the white rice KDML. Moreover, GBR of MD showed higher total anthocyanin content and greater antioxidant activity, which is consistent with the increase expression of several proteins involved in the biosynthesis of phenolic compounds and protection against oxidative stress. Red rice MD exhibits higher nutrient values compared to white rice KDML and the appropriate germination of brown rice could represent a method for improving health-related benefits. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Functional protease profiling for diagnosis of malignant disease.

Science.gov (United States)

Findeisen, Peter; Neumaier, Michael

2012-01-01

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Subgroup-specific intrinsic disorder profiles of arabidopsis NAC transcription factors

DEFF Research Database (Denmark)

Stender, Emil G.; O'Shea, Charlotte; Skriver, Karen

2015-01-01

disordered but contain short, functionally important regions with structure propensities known as molecular recognition features. Here, we analyze for NAC subgroup-specific ID patterns. Some subgroups, such as the VND subgroup implicated in secondary cell wall biosynthesis, and the NAP/SHYG subgroup have...... highly conserved ID profiles. For the stress-associated ATAF1 subgroup and the CUC/ORE1 subgroup involved in development, only sub clades have similar ID patterns. For similar ID profiles, conserved molecular recognition features and sequence motifs represent likely functional determinants of e.......g. transcriptional activation and interactions. Based on our analysis, we suggest that ID profiling of regulatory proteins in general can be used to guide identification of interaction partners of network proteins....

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

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Meinicke, Peter

2009-09-02

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. 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. 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.

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

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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.

Serum protein profile of Malaria patients through SDS-PAGE method ...

African Journals Online (AJOL)

Serum protein profile of Malaria patients through SDS-PAGE method. ... reliable method in the diagnosis of antibodies produced against Plasmodium spps. ... of malaria patients may be undertaken for study to develop possible future vaccine.

Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

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Ullal, Adeeti V; Peterson, Vanessa; Agasti, Sarit S; Tuang, Suan; Juric, Dejan; Castro, Cesar M; Weissleder, Ralph

2014-01-15

Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

TALE factors poise promoters for activation by Hox proteins.

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Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

2014-01-27

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

Bioinformatics Approach Based Research of Profile Protein Carbonic Anhydrase II Analysis as a Potential Candidate Cause Autism for The Variation of Learning Subjects Biotechnology

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Dian Eka A. F. Ningrum

2017-03-01

Full Text Available This study aims to determine the needs of learning variations on Biotechnology courses using bioinformatics approaches. One example of applied use of bioinformatics in biotechnology course is the analysis of protein profiles carbonic anhydrase II as a potential cause of autism candidate. This research is a qualitative descriptive study consisted of two phases. The first phase of the data obtained from observations of learning, student questionnaires, and questionnaires lecturer. Results from the first phase, namely the need for variations learning in Biotechnology course using bioinformatics. Collecting data on the second stage uses three webserver to predict the target protein and scientific articles. Visualization of proteins using PyMOL software. 3 based webserver which is used, the candidate of target proteins associated with autism is carbonic anhydrase II. The survey results revealed that the protein carbonic anhydrase II as a potential candidate for the cause of autism classified metaloenzim are able to bind with heavy metals. The content of heavy metals in autistic patients high that affect metabolism. This prediction of protein candidate cause autism is applied use to solve the problem in society, so that can achieve the learning outcome in biotechnology course.

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

DEFF Research Database (Denmark)

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

2015-01-01

. Aspartic and glutamic acids dominated the amino acid profile, accounting for up to 49 % of the total. Greatest seasonal differences in amino acid composition occurred in July, with leucine contributing most (22.7–26.7 %) of the observed differences. A maximal essential amino acid (EAA) score of 68......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.......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...

Protein-Based Drug-Delivery Materials

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Dave Jao

2017-05-01

Full Text Available There is a pressing need for long-term, controlled drug release for sustained treatment of chronic or persistent medical conditions and diseases. Guided drug delivery is difficult because therapeutic compounds need to survive numerous transport barriers and binding targets throughout the body. Nanoscale protein-based polymers are increasingly used for drug and vaccine delivery to cross these biological barriers and through blood circulation to their molecular site of action. Protein-based polymers compared to synthetic polymers have the advantages of good biocompatibility, biodegradability, environmental sustainability, cost effectiveness and availability. This review addresses the sources of protein-based polymers, compares the similarity and differences, and highlights characteristic properties and functionality of these protein materials for sustained and controlled drug release. Targeted drug delivery using highly functional multicomponent protein composites to guide active drugs to the site of interest will also be discussed. A systematical elucidation of drug-delivery efficiency in the case of molecular weight, particle size, shape, morphology, and porosity of materials will then be demonstrated to achieve increased drug absorption. Finally, several important biomedical applications of protein-based materials with drug-delivery function—including bone healing, antibiotic release, wound healing, and corneal regeneration, as well as diabetes, neuroinflammation and cancer treatments—are summarized at the end of this review.

Fluorogen-activating proteins: beyond classical fluorescent proteins

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Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

Effect of high-protein or normal-protein diet on weight loss, body composition, hormone, and metabolic profile in southern Brazilian women with polycystic ovary syndrome: a randomized study.

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Toscani, Mariana K; Mario, Fernanda M; Radavelli-Bagatini, Simone; Wiltgen, Denusa; Matos, Maria Cristina; Spritzer, Poli Maria

2011-11-01

The aim of the present study was to assess the effects of a high protein (HP) and a normal protein (NP) diet on patients with polycystic ovary syndrome (PCOS) and body mass index-matched controls in a sample of southern Brazilian women. This 8-week randomized trial was carried out at a university gynecological endocrinology clinic and included 18 patients with PCOS and 22 controls. Changes in weight, body composition, hormone, and metabolic profile were analyzed in women randomized to receive HP (30% protein, 40% carbohydrate, and 30% lipid) or NP (15% protein, 55% carbohydrate, and 30% lipid). The energy content was estimated for each participant at 20-25 kcal/kg current weight/day. Physical activity, blood pressure, homeostasis model assessment (HOMA) index, and fasting and 2-h glucose and insulin remained stable during the intervention in PCOS and controls, even in the presence of weight loss. There were no changes in lipid profile in either group. In contrast, body weight, body mass index (BMI), waist circumference, percent of body fat, and sum of trunk skinfolds decreased significantly after both diets in both groups. Total testosterone also decreased in PCOS and controls regardless of diet. In conclusion, calorie reduction, rather than protein content, seemed to affect body composition and hormonal profile in this short-term study. These findings emphasize the role of non-pharmacological interventions to reduce weight and ameliorate the anthropometric and clinical phenotype in PCOS.

Oxidative profiles of LDL and HDL isolated from women with preeclampsia.

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León-Reyes, G; Maida-Claros, R F; Urrutia-Medina, A X; Jorge-Galarza, E; Guzmán-Grenfell, A M; Fuentes-García, S; Medina-Navarro, R; Moreno-Eutimio, M A; Muñoz-Sánchez, J L; Hicks, J J; Torres-Ramos, Y D

2017-05-16

Oxidative stress causes biochemical changes in lipids and proteins; these changes can induce damage to the vascular endothelium and create maternal complications that are characteristic of preeclampsia. In this study, we evaluated the oxidative profile of lipoproteins isolated from women with preeclampsia. Thirty women diagnosed with preeclampsia and thirty women without preeclampsia were included in the study. Lipid-damage biomarkers, including conjugated dienes, lipohydroperoxides and malondialdehyde, were measured. The reduction of nitroblue tetrazolium, the formation of dityrosines, and the carbonylation of proteins were assessed as indicators of protein damage. The protective activity of HDL-c was evaluated by the paraoxonase-I activity present on the HDL-c particles. Serum lipid profiles were also quantified in both groups. Data were analysed using Student's t test and the Pearson correlation coefficient. Our results demonstrated in PE women evident oxidative changes in the lipids and proteins in HDL-c and LDL-c particles and the activity of the antioxidant enzyme PON-I decreased 59.9%. HDL-c exhibited self-defence, as demonstrated by the negative correlation between paraoxonase-I activity and the formation of lipohydroperoxides in HDL-c (r = -0.3755, p preeclampsia show oxidative damage to lipids and proteins. We propose an oxidative profile based on the oxidation levels indicated by each of the markers used. We also found that paraoxonase-I is inactivated in the presence of lipohydroperoxides. Antioxidant support might be helpful to reduce oxidative stress in patients with preeclampsia. Further investigations are necessary to define the association between antioxidant activities and preeclampsia.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

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Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

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Yannan Qin

2014-11-01

Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

Science.gov (United States)

Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

2013-12-01

Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient ( i.e ., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs . 252 ± 43 m, p ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly ( p ion (551.21 m/z ) of the doubly-charged peptide SLGVGFATR (454.19 m/z ) of residues 23-31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater ( p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater ( p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for

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

International Nuclear Information System (INIS)

David, M.; Lobera, A.; Legrand, E.

1984-01-01

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, alpha 1 -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 [fr

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

Energy Technology Data Exchange (ETDEWEB)

David, M; Lobera, A; Legrand, E [Fondation Bergorie, Bordeaux (France)

1984-01-01

Patients with a cancer of the upper airways on 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, alpha/sub 1/-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.

Coculture of Bifidobacterium longum and Bifidobacterium breve alters their protein expression profiles and enzymatic activities.

Science.gov (United States)

Ruiz, Lorena; Sánchez, Borja; de Los Reyes-Gavilán, Clara G; Gueimonde, Miguel; Margolles, Abelardo

2009-07-31

Some strains of the genus Bifidobacterium are probiotic bacteria commonly added to functional dairy products. The influence of coculturing Bifidobacterium longum NCIMB8809 and Bifidobacterium breve NCIMB8807 on their physiology was studied. 2DE separation of protein extracts, coupled to MS protein analysis allowed the identification of 16 proteins whose expression drastically changed when cells were grown in compartmentalized coculture, compared to monoculture. These included ribosomal proteins and proteins involved in carbohydrate metabolism, gene regulation, cell envelope biogenesis and transport processes. Significant changes in some glycoside-hydrolysing activities (beta-d-xylopyranosidase, alpha-l-arabinofuranosidase and beta-d-glucopyranosidase) were also detected. Furthermore, qRT-PCR experiments using as targets the B. breve genes clgR (transcriptional regulator) clpP1, clpP2 and clpC (chaperone- and protease-encoding genes positively regulated by clgR) supported the proteomic results, the four genes displaying a higher expression level in coculture. This study provides new insights to understand the communication among Bifidobacterium species.

AMP-activated protein kinase and type 2 diabetes.

Science.gov (United States)

Musi, Nicolas

2006-01-01

AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge, being activated in situations of high-energy phosphate depletion. Upon activation, AMPK functions to restore cellular ATP by modifying diverse metabolic pathways. AMPK is activated robustly by skeletal muscle contraction and myocardial ischemia, and may be involved in the stimulation of glucose transport and fatty acid oxidation produced by these stimuli. In liver, activation of AMPK results in enhanced fatty acid oxidation and in decreased production of glucose, cholesterol, and triglycerides. Recent studies have shown that AMPK is the cellular mediator for many of the metabolic effects of drugs such as metformin and thiazolidinediones, as well as the insulin sensitizing adipocytokines leptin and adiponectin. These data, along with evidence from studies showing that chemical activation of AMPK in vivo with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) improves blood glucose concentrations and lipid profiles, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes and other metabolic disorders.

Mass spectrometry based proteomics profiling as diagnostic tool in oncology: current status and future perspective.

Science.gov (United States)

Findeisen, Peter; Neumaier, Michael

2009-01-01

Proteomics analysis has been heralded as a novel tool for identifying new and specific biomarkers that may improve diagnosis and monitoring of various disease states. Recent years have brought a number of proteomics profiling technologies. Although proteomics profiling has resulted in the detection of disease-associated differences and modification of proteins, current proteomics technologies display certain limitations that are hampering the introduction of these new technologies into clinical laboratory diagnostics and routine applications. In this review, we summarize current advances in mass spectrometry based biomarker discovery. The promises and challenges of this new technology are discussed with particular emphasis on diagnostic perspectives of mass-spectrometry based proteomics profiling for malignant diseases.

Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

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Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

2015-05-01

Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

2-DE and MS analysis of key proteins in the adhesion of Lactobacillus plantarum, a first step toward early selection of probiotics based on bacterial biomarkers

NARCIS (Netherlands)

Izquierdo, Esther; Horvatovich, Peter; Marchioni, Eric; Aoude-Werner, Dalal; Sanz, Yolanda; Ennahar, Saied

The identification of cell components involved in probiotic activities is a challenge in current probiotic research. In this work, a new approach based on proteomics as an analytical tool for the identification of characteristic protein profiles related to adhesion to mucin as a model probiotic

Fractionation, amino acid profiles, antimicrobial and free radical scavenging activities of Citrullus lanatus seed protein.

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Dash, Priyanka; Ghosh, Goutam

2017-12-01

In the present study, a modified Osborne fractionation method was followed to isolate albumin (C alb ), globulin (C glo ), prolamin (C pro ) and glutelin (C glu ) successively from seeds of Citrullus lanatus (watermelon). This research work was undertaken to investigate the antimicrobial and antioxidant activities of isolated protein fractions of C. lanatus seed. Amino acid composition and molecular weight distribution were determined to establish their relationship with antimicrobial and antioxidant activity. Among all the fractions, C pro was found to be most effective against A. baumannii followed by C alb and C glo . The results showed that growth of inhibition of these protein fractions differ significantly from each other (p ≤ 0.05). In view of antioxidant potential, C glo exhibited strongest antioxidant capacity while C glu showed weakest antioxidant potential.

Manipulating fatty acid biosynthesis in microalgae for biofuel through protein-protein interactions.

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Jillian L Blatti

Full Text Available Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP and thioesterase (TE govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.

Mission Profile Based Sizing of IGBT Chip Area for PV Inverter Applications

DEFF Research Database (Denmark)

Shen, Yanfeng; Wang, Huai; Yang, Yongheng

2016-01-01

Maximizing the total energy generation is of importance for Photovoltaic (PV) plants. This paper proposes a method to optimize the IGBT chip area for PV inverters to minimize the annual energy loss of the active switches based on long-term operation conditions (i.e., mission profile). The design...... yearly mission profile. Simulation results are given to verify the thermal characteristics. Furthermore, a Monte Carlo based lifetime evaluation is presented to check the IGBT reliability. The proposed design method enables a reliability-oriented energy optimized sizing of active switches for PV inverter...

Effects of whole grain, fish and bilberries on serum metabolic profile and lipid transfer protein activities: a randomized trial (Sysdimet.

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Maria Lankinen

Full Text Available We studied the combined effects of wholegrain, fish and bilberries on serum metabolic profile and lipid transfer protein activities in subjects with the metabolic syndrome.Altogether 131 subjects (40-70 y, BMI 26-39 kg/m(2 with impaired glucose metabolism and features of the metabolic syndrome were randomized into three groups with 12-week periods according to a parallel study design. They consumed either: a wholegrain and low postprandial insulin response grain products, fatty fish 3 times a week, and bilberries 3 portions per day (HealthyDiet, b wholegrain and low postprandial insulin response grain products (WGED, or c refined wheat breads as cereal products (Control. Altogether 106 subjects completed the study. Serum metabolic profile was studied using an NMR-based platform providing information on lipoprotein subclasses and lipids as well as low-molecular-weight metabolites.There were no significant differences in clinical characteristics between the groups at baseline or at the end of the intervention. Mixed model analyses revealed significant changes in lipid metabolites in the HealthyDiet group during the intervention compared to the Control group. All changes reflected increased polyunsaturation in plasma fatty acids, especially in n-3 PUFAs, while n-6 and n-7 fatty acids decreased. According to tertiles of changes in fish intake, a greater increase of fish intake was associated with increased concentration of large HDL particles, larger average diameter of HDL particles, and increased concentrations of large HDL lipid components, even though total levels of HDL cholesterol remained stable.The results suggest that consumption of diet rich in whole grain, bilberries and especially fatty fish causes changes in HDL particles shifting their subclass distribution toward larger particles. These changes may be related to known protective functions of HDL such as reverse cholesterol transport and could partly explain the known protective

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

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Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

Lifestyle profile assessment in active and non-active hypertensive women

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N. R. Cavichia

2016-05-01

Full Text Available The present study aimed to evaluate the lifestyle´s profile of 30 hypertensive women practitioners and nonpractitioners of physical activity in the city of Sinop/MT. We used the questionnaire Profile of Single Lifestyle (PSL consists of three issues of the components of Nutrition (N, Physical Activity (PA, Preventive Behavior (PB, Social Networking (SN and Stress Management (SM with scores ranging from 0 to 3 points and score calculated by the sum of questions divided by the number of questions. The data were analyzed statistically with a significance of 5%. The average of the active components of PSL (N = 2,07, PA = 2,04, PB = 2,18, SN = 2,38 and SM = 2,42 and average overall score of 2,16 inactive (N = 1,73, AF = 1,33, PB = 2,40, RS = 2,02 and SN = 2,09 and average score 1,86. In the comparison between groups all components were significant different. It was concluded that the profile of the active lifestyle of hypertensive women have significantly higher values than non-active women

Characterization of Co-Cultivation of Cyanobacteria on Growth, Productions of Polysaccharides and Extracellular Proteins, Nitrogenase Activity, and Photosynthetic Activity.

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Xue, Chuizhao; Wang, Libo; Wu, Tong; Zhang, Shiping; Tang, Tao; Wang, Liang; Zhao, Quanyu; Sun, Yuhan

2017-01-01

Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers and reestablish the ecological system in soil. In general, several strains of cyanobacteria were involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity for three selected N 2 -fixing cyanobacteria, Anabaena cylindrica (B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations compared with pure culture. The highest released polysaccharides (RPS) contents were obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose were major components of CPS and RPS in all samples. Trehalose was a specific component of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280 was the dominant species in the related treatments of co-cultivation. The nitrogenase activities in all treatments exhibited a sharp rise at the late stage while a significant decrease existed when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were determined with rapid light curve, and the related parameters were estimated.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

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Kannan, Soumya; Sams, Thomas; Maury, Jérôme; Workman, Christopher T

2018-03-16

Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

Science.gov (United States)

Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

2012-01-01

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

Variance decomposition of protein profiles from antibody arrays using a longitudinal twin model

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Kato Bernet S

2011-11-01

Full Text Available Abstract Background The advent of affinity-based proteomics technologies for global protein profiling provides the prospect of finding new molecular biomarkers for common, multifactorial disorders. The molecular phenotypes obtained from studies on such platforms are driven by multiple sources, including genetic, environmental, and experimental components. In characterizing the contribution of different sources of variation to the measured phenotypes, the aim is to facilitate the design and interpretation of future biomedical studies employing exploratory and multiplexed technologies. Thus, biometrical genetic modelling of twin or other family data can be used to decompose the variation underlying a phenotype into biological and experimental components. Results Using antibody suspension bead arrays and antibodies from the Human Protein Atlas, we study unfractionated serum from a longitudinal study on 154 twins. In this study, we provide a detailed description of how the variation in a molecular phenotype in terms of protein profile can be decomposed into familial i.e. genetic and common environmental; individual environmental, short-term biological and experimental components. The results show that across 69 antibodies analyzed in the study, the median proportion of the total variation explained by familial sources is 12% (IQR 1-22%, and the median proportion of the total variation attributable to experimental sources is 63% (IQR 53-72%. Conclusion The variability analysis of antibody arrays highlights the importance to consider variability components and their relative contributions when designing and evaluating studies for biomarker discoveries with exploratory, high-throughput and multiplexed methods.

Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

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Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

2012-04-01

Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

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Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

2016-02-18

Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

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Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

2017-08-08

Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

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M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2016-11-01

The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers. Copyright © 2016 Elsevier GmbH. All rights reserved.

Integrated transcriptomic and proteomic profiling of white spruce stems during the transition from active growth to dormancy.

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Galindo González, Leonardo M; El Kayal, Walid; Ju, Chelsea J-T; Allen, Carmen C G; King-Jones, Susanne; Cooke, Janice E K

2012-04-01

In the autumn, stems of woody perennials such as forest trees undergo a transition from active growth to dormancy. We used microarray transcriptomic profiling in combination with a proteomics analysis to elucidate processes that occur during this growth-to-dormancy transition in a conifer, white spruce (Picea glauca[Moench] Voss). Several differentially expressed genes were likely associated with the developmental transition that occurs during growth cessation in the cambial zone and the concomitant completion of cell maturation in vascular tissues. Genes encoding for cell wall and membrane biosynthetic enzymes showed transcript abundance patterns consistent with completion of cell maturation, and also of cell wall and membrane modifications potentially enabling cells to withstand the harsh conditions of winter. Several differentially expressed genes were identified that encoded putative regulators of cambial activity, cell development and of the photoperiodic pathway. Reconfiguration of carbon allocation figured centrally in the tree's overwintering preparations. For example, genes associated with carbon-based defences such as terpenoids were down-regulated, while many genes associated with protein-based defences and other stress mitigation mechanisms were up-regulated. Several of these correspond to proteins that were accumulated during the growth-to-dormancy transition, emphasizing the importance of stress protection in the tree's adaptive response to overwintering. © 2011 Blackwell Publishing Ltd.

A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

NARCIS (Netherlands)

van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

1998-01-01

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper.

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Jiang, Weidong; Mao, Ying Qing; Huang, Ruochun; Duan, Chaohui; Xi, Yun; Yang, Kai; Huang, Ruo-Pan

2014-01-31

Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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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.

Protein stability and enzyme activity at extreme biological temperatures

International Nuclear Information System (INIS)

Feller, Georges

2010-01-01

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

Environmental Distribution and Diversity of Insecticidal Proteins of Bacillus thuringiensis Berliner

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Xavier, R.

2007-01-01

Full Text Available Bacillus thuringiensis Berliner based biopesticides have been successfully used world over for the control of agricultural pests and vectors of human diseases. Currently there are more than 200 B. thuringiensis strains with differing insecticidal activities are available as biocontrol agents and for developing transgenic plants. However, two major disadvantages are the development of insect resistance and high target specificity (narrow host range. Globally there is a continuous search for new B. thuringiensis strains with novel insecticidal activities. The present study aims to study the environmental distribution of B. thuringiensis and their toxic potential against insect pests. Soil and grain samples were collected from different environments and were processed by a modified acetate selection method. Initially B. thuringiensis isolates were screened on the basis of colony morphology and phase contrast microscopy for the presence of parasporal crystal inclusions. The population dynamics showed that B. thuringiensis is abundant in sericulture environment compared to other niches. Relative abundance of B. thuringiensis strains in sericulture environment shows the persistent association of B. thuringiensis with Bombyx mori (silk worm as insect pathogen. The protein profiles of the selected strains were studied by SDS-PAGE. The protein profiles of majority of B. thuringiensis isolates from grain storage facilities predominantly showing the 130 kDa and 68 kDa proteins, which is characteristics of lepidopteran active B. thuringiensis. However, one isolate BTRX-4 has 80-85 kDa protein, which is novel in that, this strain also exhibits antilepidopteran activity, which is normally presented by B. thuringiensis strains having 130 kDa and 68 kDa proteins. The protein profile of B. thuringiensis isolates from sericulture environment shows two different protein profiles. B. thuringiensis isolates BTRX-16 to BTRX-22 predominantly show 130 kDa protein

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

Science.gov (United States)

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

2003-01-01

Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

Chemical-genetic profile analysis of five inhibitory compounds in yeast.

Science.gov (United States)

Alamgir, Md; Erukova, Veronika; Jessulat, Matthew; Azizi, Ali; Golshani, Ashkan

2010-08-06

Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s). Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays. Chemical-genetic profiles provide insight into the molecular mechanism(s) of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.

Oral delivery of peptides and proteins using lipid-based drug delivery systems.

Science.gov (United States)

Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

2012-10-01

In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism by which intestinal absorption of peptides and proteins is promoted. The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two most important barriers (extensive enzymatic degradation and poor transmucosal permeability). This paper also gives a clear-cut idea about advantages and drawbacks of using different lipidic colloidal carriers ((micro)emulsions, solid lipid core particles and liposomes) for oral delivery of peptides and proteins. Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve the design and development of lipid-based DDS with the desired bioavailability and therapeutic profile.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

Science.gov (United States)

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

2009-12-01

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

A Discrete Dynamical System Approach to Pathway Activation Profiles of Signaling Cascades.

Science.gov (United States)

Catozzi, S; Sepulchre, J-A

2017-08-01

In living organisms, cascades of covalent modification cycles are one of the major intracellular signaling mechanisms, allowing to transduce physical or chemical stimuli of the external world into variations of activated biochemical species within the cell. In this paper, we develop a novel method to study the stimulus-response of signaling cascades and overall the concept of pathway activation profile which is, for a given stimulus, the sequence of activated proteins at each tier of the cascade. Our approach is based on a correspondence that we establish between the stationary states of a cascade and pieces of orbits of a 2D discrete dynamical system. The study of its possible phase portraits in function of the biochemical parameters, and in particular of the contraction/expansion properties around the fixed points of this discrete map, as well as their bifurcations, yields a classification of the cascade tiers into three main types, whose biological impact within a signaling network is examined. In particular, our approach enables to discuss quantitatively the notion of cascade amplification/attenuation from this new perspective. The method allows also to study the interplay between forward and "retroactive" signaling, i.e., the upstream influence of an inhibiting drug bound to the last tier of the cascade.

Multivariate analysis of protein profiles of metal hyperaccumulator Thlaspi caerulescens accessions.

NARCIS (Netherlands)

Tuomainen, M.H.; Nunan, N.; Lehesranta, S.J.; Tervahauta, A.I.; Hassinen, V.H.; Schat, H.; Koistinen, K.M.; Auriola, S.; McNicol, J.; Karenlampi, S.O.

2006-01-01

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyper-accumulation, we used proteomic profiling to identify differences in protein intensities among three T caerulescens

Amino acid profiles of rumen undegradable protein: a comparison between forages including cereal straws and alfalfa and their respective total mixed rations.

Science.gov (United States)

Wang, B; Jiang, L S; Liu, J X

2018-06-01

Optimizing the amino acid (AA) profile of rumen undegradable protein (RUP) can positively affect the amount of milk protein. This study was conducted to improve knowledge regarding the AA profile of rumen undegradable protein from corn stover, rice straw and alfalfa hay as well as the total mixed ratio diets (TMR) based on one of them as forage source [forage-to-concentrate ratio of 45:55 (30% of corn stover (CS), 30% of rice straw (RS), 23% of alfalfa hay (AH) and dry matter basis)]. The other ingredients in the three TMR diets were similar. The RUP of all the forages and diets was estimated by incubation for 16 hr in the rumen of three ruminally cannulated lactating cows. All residues were corrected for microbial colonization, which was necessary in determining the AA composition of RUP from feed samples using in situ method. Compared with their original AA composition, the AA pattern of forages and forage-based diets changed drastically after rumen exposure. In addition, the extent of ruminal degradation of analysed AA was not constant among the forages. The greatest individual AA degradability of alfalfa hay and corn stover was Pro, but was His of rice straw. A remarkable difference was observed between microbial attachment corrected and uncorrected AA profiles of RUP, except for alfalfa hay and His in the three forages and TMR diets. The ruminal AA degradability of cereal straws was altered compared with alfalfa hay but not for the TMR diets. In summary, the AA composition of forages and TMR-based diets changed significantly after ruminal exposure, indicating that the original AA profiles of the feed cannot represent its AA composition of RUP. The AA profile of RUP and ruminal AA degradability for corn stover and rice straw contributed to missing information in the field. © 2017 Blackwell Verlag GmbH.

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S

NARCIS (Netherlands)

van Wijnen, M.; Stam, J. G.; van't Veer, C.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

1996-01-01

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in

Protein profiles of serum, brain regions and hypophyses of pubertal ...

African Journals Online (AJOL)

The effects of dietary fumonisin B1 (FB1 ), a toxin produced mainly by Fusarium verticillioides and F. proliferatum that grow on maize worldwide, on protein profiles of serum, brain regions and hypophyses were studied in 24 male Large White weanling pigs randomly divided into four groups (n = 6). In a completely ...

Nanochemistry of protein-based delivery agents

Science.gov (United States)

Rajendran, Subin; Udenigwe, Chibuike; Yada, Rickey

2016-07-01

The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

UML Profile for Mining Process: Supporting Modeling and Simulation Based on Metamodels of Activity Diagram

Directory of Open Access Journals (Sweden)

Andrea Giubergia

2014-01-01

Full Text Available An UML profile describes lightweight extension mechanism to the UML by defining custom stereotypes, tagged values, and constraints. They are used to adapt UML metamodel to different platforms and domains. In this paper we present an UML profile for models supporting event driving simulation. In particular, we use the Arena simulation tool and we focus on the mining process domain. Profiles provide an easy way to obtain well-defined specifications, regulated by the Object Management Group (OMG. They can be used as a presimulation technique to obtain solid models for the mining industry. In this work we present a new profile to extend the UML metamodel; in particular we focus on the activity diagram. This extended model is applied to an industry problem involving loading and transportation of minerals in the field of mining process.

Antioxidant and antimicrobial activity of lecithin free egg yolk protein preparation hydrolysates obtained with digestive enzymes

Directory of Open Access Journals (Sweden)

Aleksandra Zambrowicz

2012-12-01

Full Text Available ABSTRACT:Several biological activities have now been associated with egg protein- derived peptides, including antihypertensive, antimicrobial, immunomodulatory, anticancer and antioxidantactivities, highlighting the importance of these biopeptides in human health, and disease prevention and treatment. Special attention has been given to peptides with antioxidant and antimicrobial activities as a new source of natural preservatives in food industry. In this study, the antioxidant properties of the egg-yolk protein by-product (YP hydrolysates were evaluated based on their radical scavenging capacity (DPPH, Fe2+chelating effect and ferric reducing power (FRAP. Furthermore, antimicrobial properties of obtained hydrolysates against Bacillus species were studied. The degrees (DHs of hydrolysis for 4h hydrolysates were: 19.1%, 13.5% and 13.0%, for pepsin, chymotrypsin and trypsin, respectively. Pepsin was the most effective in producing the free amino groups (1410.3 μmolGly/g. The RP-HPLC profiles of the protein hydrolysates showed differences in the hydrophobicity of the generated peptides.Trypsin hydrolysate obtained after 4h reaction demonstrated the strongest DPPH free radical scavenging activity (0.85 µmol Troloxeq/mg. Trypsin and chymotrypsin hydrolysates obtained after 4h reaction exhibited 4 times higher ferric reducing capacity than those treated bypepsin. The hydrolysis products obtained from YP exhibited significant chelating activity. The 4h trypsin hydrolysate exhibited weak antimicrobial activity against B. subtilis B3; B. cereus B512; B. cereus B 3p and B. laterosporum B6.

Probing intracellular motor protein activity using an inducible cargo trafficking assay

NARCIS (Netherlands)

L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

2010-01-01

textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

Protein Profiles for Muscle Development and Intramuscular Fat Accumulation at Different Post-Hatching Ages in Chickens.

Directory of Open Access Journals (Sweden)

Jie Liu

Full Text Available Muscle development and growth influences the efficiency of poultry meat production, and is closely related to deposition of intramuscular fat (IMF, which is crucial in meat quality. To clarify the molecular mechanisms underlying muscle development and IMF deposition in chickens, protein expression profiles were examined in the breast muscle of Beijing-You chickens at ages 1, 56, 98 and 140 days, using isobaric tags for relative and absolute quantification (iTRAQ. Two hundred and four of 494 proteins were expressed differentially. The expression profile at day 1 differed greatly from those at day 56, 98 and 140. KEGG pathway analysis of differential protein expression from pair-wise comparisons (day 1 vs. 56; 56 vs. 98; 98 vs. 140, showed that the fatty acid degradation pathway was more active during the stage from day 1 to 56 than at other periods. This was consistent with the change in IMF content, which was highest at day 1 and declined dramatically thereafter. When muscle growth was most rapid (days 56-98, pathways involved in muscle development were dominant, including hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, tight junctions and focal adhesion. In contrast with hatchlings, the fatty acid degradation pathway was downregulated from day 98 to 140, which was consistent with the period for IMF deposition following rapid muscle growth. Changes in some key specific proteins, including fast skeletal muscle troponin T isoform, aldehyde dehydrogenase 1A1 and apolipoprotein A1, were verified by Western blotting, and could be potential biomarkers for IMF deposition in chickens. Protein-protein interaction networks showed that ribosome-related functional modules were clustered in all three stages. However, the functional module involved in the metabolic pathway was only clustered in the first stage (day 1 vs. 56. This study improves our understanding of the molecular mechanisms underlying muscle development and IMF

Optimization of translation profiles enhances protein expression and solubility.

Directory of Open Access Journals (Sweden)

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.

Domain-oriented functional analysis based on expression profiling

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Greene Jonathan

2002-10-01

Full Text Available Abstract Background Co-regulation of genes may imply involvement in similar biological processes or related function. Many clusters of co-regulated genes have been identified using microarray experiments. In this study, we examined co-regulated gene families using large-scale cDNA microarray experiments on the human transcriptome. Results We present a simple model, which, for each probe pair, distills expression changes into binary digits and summarizes the expression of multiple members of a gene family as the Family Regulation Ratio. The set of Family Regulation Ratios for each protein family across multiple experiments is called a Family Regulation Profile. We analyzed these Family Regulation Profiles using Pearson Correlation Coefficients and derived a network diagram portraying relationships between the Family Regulation Profiles of gene families that are well represented on the microarrays. Our strategy was cross-validated with two randomly chosen data subsets and was proven to be a reliable approach. Conclusion This work will help us to understand and identify the functional relationships between gene families and the regulatory pathways in which each family is involved. Concepts presented here may be useful for objective clustering of protein functions and deriving a comprehensive protein interaction map. Functional genomic approaches such as this may also be applicable to the elucidation of complex genetic regulatory networks.

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

OpenAIRE

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

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.

On active current selection for Lagrangian profilers

DEFF Research Database (Denmark)

Jouffroy, Jerome; Zhou, Qiuyang; Zielinski, Oliver

2013-01-01

simple and computationally-efficient control strategies to actively select and use ocean currents so that a profiler can autonomously reach a desired destination. After briefly presenting a typical profiler and possible mechanical modifications for a coastal environment, we introduce simple mathematical...... models for the profiler and the currents it will use. We then present simple feedback controllers that, using the direction of the currents and taking into account the configuration of the environment (coastal or deep-sea), is able to steer the profiler to any desired horizontal location. To illustrate...

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation

Science.gov (United States)

Vandame, Pauline; Spriet, Corentin; Trinel, Dave; Gelaude, Armance; Caillau, Katia; Bompard, Coralie; Biondi, Emanuele; Bodart, Jean-François

2014-01-01

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells. PMID:25485503

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation.

Science.gov (United States)

Vandame, Pauline; Spriet, Corentin; Trinel, Dave; Gelaude, Armance; Caillau, Katia; Bompard, Coralie; Biondi, Emanuele; Bodart, Jean-François

2014-01-01

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.

Nanochemistry of protein-based delivery agents

Directory of Open Access Journals (Sweden)

Subin R.C.K. Rajendran

2016-07-01

Full Text Available The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

Chemical-genetic profile analysis of five inhibitory compounds in yeast

Directory of Open Access Journals (Sweden)

Alamgir Md

2010-08-01

Full Text Available Abstract Background Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s. Results Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays. Conclusion Chemical-genetic profiles provide insight into the molecular mechanism(s of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.

Structure-based drug design for G protein-coupled receptors.

Science.gov (United States)

Congreve, Miles; Dias, João M; Marshall, Fiona H

2014-01-01

Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed. © 2014 Elsevier B.V. All rights reserved.

RELATIONSHIPS BETWEEN PHYSICAL EDUCATION STUDENTS' MOTIVATIONAL PROFILES, ENJOYMENT, STATE ANXIETY, AND SELF-REPORTED PHYSICAL ACTIVITY

Directory of Open Access Journals (Sweden)

Sami Yli-Piipari

2009-09-01

Full Text Available The purpose of this study was to analyze motivational profiles based on the self-determination theory (Deci and Ryan, 2000 and how these profiles are related to physical education students' enjoyment, state anxiety, and physical activity. The participants, 429 sixth grade students (girls = 216; boys = 213 completed SMS, Sport Enjoyment Scale, PESAS, and Physical Activity Scale. Cluster analyses identified two motivational profiles: 1 the "High motivation profile", in which the students had high intrinsic and extrinsic motivation, and low levels of amotivation, and 2 the "Low motivation profile", in which the students had low intrinsic and extrinsic motivation, and low levels of amotivation. The students in the first cluster enjoyed physical education more and were physically more active. The results revealed that students may be motivated towards physical education lessons both intrinsically and extrinsically, and still experience enjoyment in physical education.

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

Science.gov (United States)

Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

2014-01-01

Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

Serum protein profiling and proteomics in autistic spectrum disorder using magnetic bead-assisted mass spectrometry.

Science.gov (United States)

Taurines, Regina; Dudley, Edward; Conner, Alexander C; Grassl, Julia; Jans, Thomas; Guderian, Frank; Mehler-Wex, Claudia; Warnke, Andreas; Gerlach, Manfred; Thome, Johannes

2010-04-01

The pathophysiology of autistic spectrum disorder (ASD) is not fully understood and there are no diagnostic or predictive biomarkers. Proteomic profiling has been used in the past for biomarker research in several non-psychiatric and psychiatric disorders and could provide new insights, potentially presenting a useful tool for generating such biomarkers in autism. Serum protein pre-fractionation with C8-magnetic beads and protein profiling by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-ToF-MS) were used to identify possible differences in protein profiles in patients and controls. Serum was obtained from 16 patients (aged 8-18) and age-matched controls. Three peaks in the MALDI-ToF-MS significantly differentiated the ASD sample from the control group. Sub-grouping the ASD patients into children with and without comorbid Attention Deficit and Hyperactivity Disorder, ADHD (ASD/ADHD+ patients, n = 9; ASD/ADHD- patients, n = 7), one peak distinguished the ASD/ADHD+ patients from controls and ASD/ADHD- patients. Our results suggest that altered protein levels in peripheral blood of patients with ASD might represent useful biomarkers for this devastating psychiatric disorder.

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

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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.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-01-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. This study illustrates the use of the OAD in three related areas. Section I illustrates the separation of four free amino acids using cation-exchange chromatography. Detection by coupling the OAD to a refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (UV) for tyrosine and phenylalanine allows the calculation of enantiomeric (D/L) ratios of these amino acids without physical separation. Specific rotations of these four amino acids are also reported. Section II illustrates the separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/UV. Section III illustrates the RP-HPLC separation of conformers of soybean trypsin inhibitor. Detection by OA/UV provides insights from the chromatogram unavailable for UV absorbance detection alone. In addition, identification of impurities is simplified with OA/UV. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation.

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

Directory of Open Access Journals (Sweden)

Daniel Thomas

Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

The Human Pancreas Proteome Defined by Transcriptomics and Antibody-Based Profiling

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Fagerberg, Linn; Hallström, Björn M.; Schwenk, Jochen M.; Uhlén, Mathias; Korsgren, Olle; Lindskog, Cecilia

2014-01-01

The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects. PMID:25546435

Profiles of Nature Exposure and Outdoor Activities Associated With Occupational Well-Being Among Employees

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Hyvönen, Katriina; Törnroos, Kaisa; Salonen, Kirsi; Korpela, Kalevi; Feldt, Taru; Kinnunen, Ulla

2018-01-01

This research addresses the profiles of nature exposure and outdoor activities in nature among Finnish employees (N = 783). The profiles were formed on the bases of nature exposure at work and the frequency and type of outdoor activities in nature engaged in during leisure time. The profiles were investigated in relation to work engagement and burnout. The latent profile analysis identified a five-class solution as the best model: High exposure (8%), Versatile exposure (22%), Unilateral exposure (38%), Average exposure (13%), and Low exposure (19%). An Analysis of Covariance (ANCOVA) was conducted for each well-being outcome in order to evaluate how the identified profiles related to occupational well-being. Participants with a High, Versatile, or Unilateral exposure profile reported significantly higher work engagement in the dimensions of vigor and dedication than did the participants with a Low exposure profile. The participants with the High exposure profile also reported lower burnout in the dimensions of cynicism and professional inadequacy than the participants with the Low exposure profile. Nature exposure during the workday and leisure time is an under researched but important aspect in promoting occupational well-being. PMID:29867699

Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling.

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Henze, Andrea; Aumer, Franziska; Grabner, Arthur; Raila, Jens; Schweigert, Florian J

2011-10-01

Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

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Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

2008-01-01

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

Directory of Open Access Journals (Sweden)

Inger Lindin

2014-03-01

Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

Novel Indole-based Tambjamine-Analogues Induce Apoptotic Lung Cancer Cell Death through p38 Mitogen-Activated Protein Kinase Activation.

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Manuel-Manresa, Pilar; Korrodi-Gregório, Luís; Hernando, Elsa; Villanueva, Alberto; Martínez-García, David; Rodilla, Ananda M; Ramos, Ricard; Fardilha, Margarida; Moya, Juan; Quesada, Roberto; Soto-Cerrato, Vanessa; Pérez-Tomás, Ricardo

2017-07-01

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5 /survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer. Mol Cancer Ther; 16(7); 1224-35. ©2017 AACR . ©2017 American Association for Cancer Research.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

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Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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Reinert, Line; Shi, B; Nandi, S

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....

Effect of plant proteins and crystalline amino acid supplementation on postprandial plasma amino acid profiles and metabolic response in rainbow trout (Oncorhynchus mykiss)

DEFF Research Database (Denmark)

Rolland, Marine; Larsen, Bodil Katrine; Holm, Jørgen

2015-01-01

.75 % of their body mass with a diet based on either (1) fish meal (FM), (2) pea protein concentrate (PPC), or (3) pea protein concentrate supplemented with histidine, lysine, methionine and threonine (PPC+) to mimic FM AA profile. The specific dynamic action and nitrogen quotient (NQ) were calculated for 48 h....... The strongest effect was observed for methionine, presenting threefold higher concentrations at peak time for PPC+ compared to FM (297.0 +/- A 77.0 and 131.8 +/- A 39.0 nmol ml(-1), respectively). The differences in AA availability and metabolic profile in the pea diets compared to the FM diet were believed...

Protein pathway activation associated with sustained virologic response in patients with chronic hepatitis C treated with pegylated interferon (PEG-IFN) and ribavirin (RBV).

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Younossi, Zobair M; Limongi, Dolores; Stepanova, Maria; Pierobon, Mariaelena; Afendy, Arian; Mehta, Rohini; Baranova, Ancha; Liotta, Lance; Petricoin, Emanuel

2011-02-04

Only half of chronic hepatitis C (CH-C) patients treated with pegylated interferon and ribavirin (PEG-IFN+RBV) achieve sustained virologic response) SVR. In addition to known factors, we postulated that activation of key protein signaling networks in the peripheral blood mononuclear cells (PBMCs) may contribute to SVR due to inherent patient-specific basal immune cell signaling architecture. In this study, we included 92 patients with CH-C. PBMCs were collected while patients were not receiving treatment and used for phosphoprotein-based network profiling. Patients received a full course of PEG-IFN+RBV with overall SVR of 55%. From PBMC, protein lysates were extracted and then used for Reverse Phase Protein Microarray (RPMA) analysis, which quantitatively measured the levels of cytokines and activation levels of 25 key protein signaling molecules involved in immune cell regulation and interferon alpha signaling. Regression models for predicting SVR were generated by stepwise bidirectional selection. Both clinical-laboratory and RPMA parameters were used as predictor variables. Model accuracies were estimated using 10-fold cross-validation. Our results show that by comparing patients who achieved SVR to those who did not, phosphorylation levels of 6 proteins [AKT(T308), JAK1(Y1022/1023), p70 S6 Kinase (S371), PKC zeta/lambda(T410/403), TYK2(Y1054/1055), ZAP-70(Y319)/Syk(Y352)] and overall levels of 6 unmodified proteins [IL2, IL10, IL4, IL5, TNF-alpha, CD5L] were significantly different (P < 0.05). For SVR, the model based on a combination of clinical and proteome parameters was developed, with an AUC = 0.914, sensitivity of 92.16%, and specificity of 85.0%. This model included the following parameters: viral genotype, previous treatment status, BMI, phosphorylated states of STAT2, AKT, LCK, and TYK2 kinases as well as steady state levels of IL4, IL5, and TNF-alpha. In conclusion, SVR could be predicted by a combination of clinical, cytokine, and protein signaling

Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection.

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Kandhavelu, Jeyalakshmi; Demonte, Naveen Luke; Namperumalsamy, Venkatesh Prajna; Prajna, Lalitha; Thangavel, Chitra; Jayapal, Jeya Maheshwari; Kuppamuthu, Dharmalingam

2017-01-30

Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense

Relationships between physical education students' motivational profiles, enjoyment, state anxiety, and self-reported physical activity.

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Yli-Piipari, Sami; Watt, Anthony; Jaakkola, Timo; Liukkonen, Jarmo; Nurmi, Jari-Erik

2009-01-01

The purpose of this study was to analyze motivational profiles based on the self-determination theory (Deci and Ryan, 2000) and how these profiles are related to physical education students' enjoyment, state anxiety, and physical activity. The participants, 429 sixth grade students (girls = 216; boys = 213) completed SMS, Sport Enjoyment Scale, PESAS, and Physical Activity Scale. Cluster analyses identified two motivational profiles: 1) the "High motivation profile", in which the students had high intrinsic and extrinsic motivation, and low levels of amotivation, and 2) the "Low motivation profile", in which the students had low intrinsic and extrinsic motivation, and low levels of amotivation. The students in the first cluster enjoyed physical education more and were physically more active. The results revealed that students may be motivated towards physical education lessons both intrinsically and extrinsically, and still experience enjoyment in physical education. Key pointsTWO MOTIVATIONAL PROFILES WERE REVEALED: 1) the "High motivation profile", in which the students had high intrinsic and extrinsic motivation, and low levels of amotivation, and 2) the "Low motivation profile", in which the students had low intrinsic and extrinsic motivation, and low levels of amotivation.The students in the first profile enjoyed physical education more and were physically more active than the students in the second profile.Moreover, the representatives of the "High motivation profile "experienced greater anxiety toward physical education than the representatives of the "Low motivation profile"These findings raised an interesting question whether students engaging in physical education benefit more from the presence of both self-determined and non-self-determined forms of motivation, or are the benefits higher if students are primarily self-determined?

Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

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Peng Liu

2015-01-01

Full Text Available A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry.

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Koc, Anna; Cañuelo, Ana; Garcia-Reyes, Juan F; Molina-Diaz, Antonio; Trojanowicz, Marek

2012-06-01

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study of protein and metabolic profile of sugarcane workers

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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)

Study of protein and metabolic profile of sugarcane workers

International Nuclear Information System (INIS)

Polachini, G.M.; Tajara, E.H.; Santos, U.P.; Zeri, A.C.M.; Paes Leme, A.F.

2012-01-01

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)

Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles.

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Mota, Rita; Pereira, Sara B; Meazzini, Marianna; Fernandes, Rui; Santos, Arlete; Evans, Caroline A; De Philippis, Roberto; Wright, Phillip C; Tamagnini, Paula

2015-04-29

The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers. Copyright © 2015. Published by Elsevier B.V.

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

International Nuclear Information System (INIS)

Liu, Chih-Wei; Bramer, Lisa; Computational Modeling); Webb-Robertson, Bobbie-Jo; Computational Modeling); Waugh, Kathleen; Rewers, Marian J.; Zhang, Qibin; Biochemistry)

2017-01-01

We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured) longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.

Nosocomial klebsiella infection in neonates in a tertiary care hospital: Protein profile by SDS-page and klebocin typing as epidemiological markers

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Malik A

2003-01-01

Full Text Available PURPOSE: To find out the prevalence of Klebsiella in hospital acquired neonatal infections in a tertiary care set up and to evaluate the role of klebocin typing and protein profile by SDS-PAGE in epidemiological typing of the isolates. METHODS: Hospital born neonates transferred to the neonatal unit after birth and available in the unit 48 hours later comprised the study group. Two hundred and three neonates were found eligible for inclusion in the study. Repeated blood cultures, other relevant clinical specimens and environmental samples were collected and identified according to the standard techniques. Isolated clinical and environmental Klebsiella pneumoniae strains were subjected to klebocin typing and protein profiling by SDS-PAGE at regular intervals. RESULTS: Multi drug resistant K. pneumoniae were the commonest organism isolated in 30 neonates leading to the incidence of Klebsiella nosocomial infection to be 14.7%. Klebocin typing of the K. pneumoniae isolates showed four patterns with type 312 being the commonest (43.4%. Whole cell protein analysis by SDS-PAGE of K. pneumoniae isolates revealed four types of banding pattern. Analysis of the typing method showed that the typeability and reproducibility of klebocin was 83.3% and 73.3% respectively whereas typeability and reproducibility of SDS-PAGE was 100%. CONCLUSIONS: Based on the present study it is concluded that SDS-PAGE typing method is better than klebocin typing in neonatal nosocomial infection. It is also suggested that protein profile by SDS-PAGE may be used as a tool for epidemiological typing of Klebsiella pneumoniae isolates in laboratories where genomic based molecular typing technique is not available.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

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Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

Science.gov (United States)

McGinley, Cian; Bishop, David J

2016-12-01

McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βm in vitro ), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βm in vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O 2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

Applying ligands profiling using multiple extended electron distribution based field templates and feature trees similarity searching in the discovery of new generation of urea-based antineoplastic kinase inhibitors.

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Eman M Dokla

Full Text Available This study provides a comprehensive computational procedure for the discovery of novel urea-based antineoplastic kinase inhibitors while focusing on diversification of both chemotype and selectivity pattern. It presents a systematic structural analysis of the different binding motifs of urea-based kinase inhibitors and the corresponding configurations of the kinase enzymes. The computational model depends on simultaneous application of two protocols. The first protocol applies multiple consecutive validated virtual screening filters including SMARTS, support vector-machine model (ROC = 0.98, Bayesian model (ROC = 0.86 and structure-based pharmacophore filters based on urea-based kinase inhibitors complexes retrieved from literature. This is followed by hits profiling against different extended electron distribution (XED based field templates representing different kinase targets. The second protocol enables cancericidal activity verification by using the algorithm of feature trees (Ftrees similarity searching against NCI database. Being a proof-of-concept study, this combined procedure was experimentally validated by its utilization in developing a novel series of urea-based derivatives of strong anticancer activity. This new series is based on 3-benzylbenzo[d]thiazol-2(3H-one scaffold which has interesting chemical feasibility and wide diversification capability. Antineoplastic activity of this series was assayed in vitro against NCI 60 tumor-cell lines showing very strong inhibition of GI(50 as low as 0.9 uM. Additionally, its mechanism was unleashed using KINEX™ protein kinase microarray-based small molecule inhibitor profiling platform and cell cycle analysis showing a peculiar selectivity pattern against Zap70, c-src, Mink1, csk and MeKK2 kinases. Interestingly, it showed activity on syk kinase confirming the recent studies finding of the high activity of diphenyl urea containing compounds against this kinase. Allover, the new series

Protein abundance profiling of the Escherichia coli cytosol

DEFF Research Database (Denmark)

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 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...

Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

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Zorzi, Giovanni K; Párraga, Jenny E; Seijo, Begoña; Sanchez, Alejandro

2015-11-01

Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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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.

Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

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Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

2017-07-01

Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

A novel engineered VEGF blocker with an excellent pharmacokinetic profile and robust anti-tumor activity

International Nuclear Information System (INIS)

Liu, Lily; Yu, Haijia; Huang, Xin; Tan, Hongzhi; Li, Song; Luo, Yan; Zhang, Li; Jiang, Sumei; Jia, Huifeng; Xiong, Yao; Zhang, Ruliang; Huang, Yi; Chu, Charles C; Tian, Wenzhi

2015-01-01

Relatively poor penetration and retention in tumor tissue has been documented for large molecule drugs including therapeutic antibodies and recombinant immunoglobulin constant region (Fc)-fusion proteins due to their large size, positive charge, and strong target binding affinity. Therefore, when designing a large molecular drug candidate, smaller size, neutral charge, and optimal affinity should be considered. We engineered a recombinant protein by molecular engineering the second domain of VEGFR1 and a few flanking residues fused with the Fc fragment of human IgG1, which we named HB-002.1. This recombinant protein was extensively characterized both in vitro and in vivo for its target-binding and target-blocking activities, pharmacokinetic profile, angiogenesis inhibition activity, and anti-tumor therapeutic efficacy. HB-002.1 has a molecular weight of ~80 kDa, isoelectric point of ~6.7, and an optimal target binding affinity of <1 nM. The pharmacokinetic profile was excellent with a half-life of 5 days, maximal concentration of 20.27 μg/ml, and area under the curve of 81.46 μg · days/ml. When tested in a transgenic zebrafish embryonic angiogenesis model, dramatic inhibition in angiogenesis was exhibited by a markedly reduced number of subintestinal vessels. When tested for anti-tumor efficacy, HB-002.1 was confirmed in two xenograft tumor models (A549 and Colo-205) to have a robust tumor killing activity, showing a percentage of inhibition over 90% at the dose of 20 mg/kg. Most promisingly, HB-002.1 showed a superior therapeutic efficacy compared to bevacizumab in the A549 xenograft model (tumor inhibition: 84.7% for HB-002.1 versus 67.6% for bevacizumab, P < 0.0001). HB-002.1 is a strong angiogenesis inhibitor that has the potential to be a novel promising drug for angiogenesis-related diseases such as tumor neoplasms and age-related macular degeneration

Accelerating Information Retrieval from Profile Hidden Markov Model Databases.

Science.gov (United States)

Tamimi, Ahmad; Ashhab, Yaqoub; Tamimi, Hashem

2016-01-01

Profile Hidden Markov Model (Profile-HMM) is an efficient statistical approach to represent protein families. Currently, several databases maintain valuable protein sequence information as profile-HMMs. There is an increasing interest to improve the efficiency of searching Profile-HMM databases to detect sequence-profile or profile-profile homology. However, most efforts to enhance searching efficiency have been focusing on improving the alignment algorithms. Although the performance of these algorithms is fairly acceptable, the growing size of these databases, as well as the increasing demand for using batch query searching approach, are strong motivations that call for further enhancement of information retrieval from profile-HMM databases. This work presents a heuristic method to accelerate the current profile-HMM homology searching approaches. The method works by cluster-based remodeling of the database to reduce the search space, rather than focusing on the alignment algorithms. Using different clustering techniques, 4284 TIGRFAMs profiles were clustered based on their similarities. A representative for each cluster was assigned. To enhance sensitivity, we proposed an extended step that allows overlapping among clusters. A validation benchmark of 6000 randomly selected protein sequences was used to query the clustered profiles. To evaluate the efficiency of our approach, speed and recall values were measured and compared with the sequential search approach. Using hierarchical, k-means, and connected component clustering techniques followed by the extended overlapping step, we obtained an average reduction in time of 41%, and an average recall of 96%. Our results demonstrate that representation of profile-HMMs using a clustering-based approach can significantly accelerate data retrieval from profile-HMM databases.

EnzDP: improved enzyme annotation for metabolic network reconstruction based on domain composition profiles.

Science.gov (United States)

Nguyen, Nam-Ninh; Srihari, Sriganesh; Leong, Hon Wai; Chong, Ket-Fah

2015-10-01

Determining the entire complement of enzymes and their enzymatic functions is a fundamental step for reconstructing the metabolic network of cells. High quality enzyme annotation helps in enhancing metabolic networks reconstructed from the genome, especially by reducing gaps and increasing the enzyme coverage. Currently, structure-based and network-based approaches can only cover a limited number of enzyme families, and the accuracy of homology-based approaches can be further improved. Bottom-up homology-based approach improves the coverage by rebuilding Hidden Markov Model (HMM) profiles for all known enzymes. However, its clustering procedure relies firmly on BLAST similarity score, ignoring protein domains/patterns, and is sensitive to changes in cut-off thresholds. Here, we use functional domain architecture to score the association between domain families and enzyme families (Domain-Enzyme Association Scoring, DEAS). The DEAS score is used to calculate the similarity between proteins, which is then used in clustering procedure, instead of using sequence similarity score. We improve the enzyme annotation protocol using a stringent classification procedure, and by choosing optimal threshold settings and checking for active sites. Our analysis shows that our stringent protocol EnzDP can cover up to 90% of enzyme families available in Swiss-Prot. It achieves a high accuracy of 94.5% based on five-fold cross-validation. EnzDP outperforms existing methods across several testing scenarios. Thus, EnzDP serves as a reliable automated tool for enzyme annotation and metabolic network reconstruction. Available at: www.comp.nus.edu.sg/~nguyennn/EnzDP .

Self-assembly of protein-based biomaterials initiated by titania nanotubes.

Science.gov (United States)

Forstater, Jacob H; Kleinhammes, Alfred; Wu, Yue

2013-12-03

Protein-based biomaterials are a promising strategy for creating robust highly selective biocatalysts. The assembled biomaterials must sufficiently retain the near-native structure of proteins and provide molecular access to catalytically active sites. These requirements often exclude the use of conventional assembly techniques, which rely on covalent cross-linking of proteins or entrapment within a scaffold. Here we demonstrate that titania nanotubes can initiate and template the self-assembly of enzymes, such as ribonuclease A, while maintaining their catalytic activity. Initially, the enzymes form multilayer thick ellipsoidal aggregates centered on the nanotube surface; subsequently, these nanosized entities assemble into a micrometer-sized enzyme material that has enhanced enzymatic activity and contains as little as 0.1 wt % TiO2 nanotubes. This phenomenon is uniquely associated with the active anatase (001)-like surface of titania nanotubes and does not occur on other anatase nanomaterials, which contain significantly fewer undercoordinated Ti surface sites. These findings present a nanotechnology-enabled mechanism of biomaterial growth and open a new route for creating stable protein-based biomaterials and biocatalysts without the need for chemical modification.

Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid

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Berle Magnus

2011-05-01

Full Text Available Abstract Background The mechanisms behind formation and filling of intracranial arachnoid cysts (AC are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1 Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2 Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF and plasma. Methods AC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM initiated detection and sequence analysis. Results We did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC. In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that

DNA-Catalytically Active Gold Nanoparticle Conjugates-Based Colorimetric Multidimensional Sensor Array for Protein Discrimination.

Science.gov (United States)

Wei, Xiangcong; Chen, Zhengbo; Tan, Lulu; Lou, Tianhong; Zhao, Yan

2017-01-03

A series of single-strand oligonucleotides functionalized catalytically active gold nanoparticle (AuNPs) as nonspecific receptors have been designed to build a protein sensing array. We take advantage of the correlation between the catalytic activity and the exposed surface area of AuNPs, i.e., DNA-proteins interactions mask the surface area of AuNPs, leading to poor catalytic performance of AuNPs. As the number of DNA-bound proteins increases, the surfaces of AuNPs become more masked; thus, the time of 4- nitrophenol/NaBH 4 reaction for color change (yellow → colorless) of the solution increases. Taking advantage of three nonspecific SH-labeled DNA sequences (A15, C15, and T15) as array sensing elements and the color-change time (CCT) of the solution as signal readout, colorimetric response patterns can be obtained on the array and identified via linear discriminant analysis (LDA). Eleven proteins have been completely distinguished with 100% accuracy with the naked eye at the 30 nM level. Remarkably, two similar proteins (bovine serum albumin and human serum albumin), two different proteins (bovine serum albumin and concanavalin) at the same concentration, and the mixtures of the two proteins with different molar ratios have been discriminated with 100%. The practicability of this sensor array is further validated by high accuracy (100%) identification of 11 proteins in human serum samples.

Physicochemical, sensory attributes and protein profile by SDS-PAGE of beef sausage substituted with texturized vegetable protein

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Hidayat, B.T.,

2017-08-01

Full Text Available The effect of texturized vegetable protein (TVP on the quality of beef sausages was investigated in this research. Several formulations which replaced by beef meat with TVP ranging from 10-20% w/w were investigated for their physical, chemical, sensory properties and also protein profile by SDS-PAGE. The addition of TVP concentration significantly influences physicochemical characteristics e.g. water, fat content, the color parameter (L and b value, WHC, Texture (Hardness and cooking yield (P<0.05. The protein profile also influenced by the addition of TVP in beef sausage formula. Higher substitution of meat with TVP will increase the water and will decrease fat content significantly (P<0.05. The highest water content is 40% TVP (64.02 ± 1.15% where the lowest water content is control (61.29 ± 1.88%. The highest fat content is control (12.16 ± 1.87% where the highest fat content is (8.53 ± 2.09%. For the physicochemical properties, e.g. L* and b* value, WHC and Cooking yield will increase during the substitution of meat with TVP in sausage products (P<0.05. The hardness will decrease during the substitution of meat with TVP in sausage products (P<0.05. Sensory results indicated that sensory attributed of beef sausage showed good acceptance until 30% of TVP substitution.

Liposome-based Formulation for Intracellular Delivery of Functional Proteins

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Benoît Chatin

2015-01-01

Full Text Available The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal enzyme and the anti-cytokeratin8 (K8 antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium-tren-cholesterol (BGTC (bis (guanidinium-tren-cholesterol combined to the colipid dioleoyl phosphatidylethanolamine (DOPE (dioleoyl phosphatidylethanolamine was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

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Xiaolin Wu

Full Text Available Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs, mainly mannose-binding lectin (agglutinin, exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE. Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

Hidden Markov models for the activity profile of terrorist groups

OpenAIRE

Raghavan, Vasanthan; Galstyan, Aram; Tartakovsky, Alexander G.

2012-01-01

The main focus of this work is on developing models for the activity profile of a terrorist group, detecting sudden spurts and downfalls in this profile, and, in general, tracking it over a period of time. Toward this goal, a $d$-state hidden Markov model (HMM) that captures the latent states underlying the dynamics of the group and thus its activity profile is developed. The simplest setting of $d=2$ corresponds to the case where the dynamics are coarsely quantized as Active and Inactive, re...

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

DEFF Research Database (Denmark)

Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

Utilizing knowledge base of amino acids structural neighborhoods to predict protein-protein interaction sites.

Science.gov (United States)

Jelínek, Jan; Škoda, Petr; Hoksza, David

2017-12-06

Protein-protein interactions (PPI) play a key role in an investigation of various biochemical processes, and their identification is thus of great importance. Although computational prediction of which amino acids take part in a PPI has been an active field of research for some time, the quality of in-silico methods is still far from perfect. We have developed a novel prediction method called INSPiRE which benefits from a knowledge base built from data available in Protein Data Bank. All proteins involved in PPIs were converted into labeled graphs with nodes corresponding to amino acids and edges to pairs of neighboring amino acids. A structural neighborhood of each node was then encoded into a bit string and stored in the knowledge base. When predicting PPIs, INSPiRE labels amino acids of unknown proteins as interface or non-interface based on how often their structural neighborhood appears as interface or non-interface in the knowledge base. We evaluated INSPiRE's behavior with respect to different types and sizes of the structural neighborhood. Furthermore, we examined the suitability of several different features for labeling the nodes. Our evaluations showed that INSPiRE clearly outperforms existing methods with respect to Matthews correlation coefficient. In this paper we introduce a new knowledge-based method for identification of protein-protein interaction sites called INSPiRE. Its knowledge base utilizes structural patterns of known interaction sites in the Protein Data Bank which are then used for PPI prediction. Extensive experiments on several well-established datasets show that INSPiRE significantly surpasses existing PPI approaches.

Effects of nutrient profiling and price changes based on NuVal® scores on food purchasing in an online experimental supermarket.

Science.gov (United States)

Epstein, Leonard H; Finkelstein, Eric A; Katz, David L; Jankowiak, Noelle; Pudlewski, Corrin; Paluch, Rocco A

2016-08-01

The goal of the present study was to apply experimental economic methods in an online supermarket to examine the effects of nutrient profiling, and differential pricing based on the nutrient profile, on the overall diet quality, energy and macronutrients of the foods purchased, and diet cost. Participants were provided nutrient profiling scores or price adjustments based on nutrient profile scores while completing a hypothetical grocery shopping task. Prices of foods in the top 20 % of nutrient profiling scores were reduced (subsidized) by 25 % while those in the bottom 20 % of scores were increased (taxed) by 25 %. We evaluated the independent and interactive effects of nutrient profiling or price adjustments on overall diet quality of foods purchased as assessed by the NuVal® score, energy and macronutrients purchased and diet cost in a 2×2 factorial design. A large (>10 000 food items) online experimental supermarket in the USA. Seven hundred and eighty-one women. Providing nutrient profiling scores improved overall diet quality of foods purchased. Price changes were associated with an increase in protein purchased, an increase in energy cost, and reduced carbohydrate and protein costs. Price changes and nutrient profiling combined were associated with no unique benefits beyond price changes or nutrient profiling alone. Providing nutrient profile score increased overall NuVal® score without a reduction in energy purchased. Combining nutrient profiling and price changes did not show an overall benefit to diet quality and may be less useful than nutrient profiling alone to consumers who want to increase overall diet quality of foods purchased.

Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria.

Science.gov (United States)

Pavankumar, Asalapuram R; Ayyappasamy, Sudalaiyadum Perumal; Sankaran, Krishnan

2012-03-01

Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.

Protein and Amino Acid Profile of Filial Etawah Crossbred and Castrated Filial Boer Crossbred Goat Meat

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Hari Purnomo

2012-03-01

Full Text Available The aim of this study was to know the protein content and amino acid profile of filial Etawah and castrated Boer goat meat. The results were expected to be used as information about protein content and amino acid composition of filial Etawah and filial castrated Boer goat meat and  as a reference for further experiment about different livestock. The material of the research were loin meat, front  and back thigh of filial Etawah and filial castrated Boer goat meat. Data were analysed with t-test. The results showed that castrated filial Boer goat meat had significantly higher protein content  and 7 essensial amino acids namely lysine, leucine, arginine, phenylalanine, isoleucine, valine and histidine compared to the one from filial Etawah goat meat. Key words: protein, amino acid profiles, goat  meat

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Science.gov (United States)

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Active Learning Strategies for Phenotypic Profiling of High-Content Screens.

Science.gov (United States)

Smith, Kevin; Horvath, Peter

2014-06-01

High-content screening is a powerful method to discover new drugs and carry out basic biological research. Increasingly, high-content screens have come to rely on supervised machine learning (SML) to perform automatic phenotypic classification as an essential step of the analysis. However, this comes at a cost, namely, the labeled examples required to train the predictive model. Classification performance increases with the number of labeled examples, and because labeling examples demands time from an expert, the training process represents a significant time investment. Active learning strategies attempt to overcome this bottleneck by presenting the most relevant examples to the annotator, thereby achieving high accuracy while minimizing the cost of obtaining labeled data. In this article, we investigate the impact of active learning on single-cell-based phenotype recognition, using data from three large-scale RNA interference high-content screens representing diverse phenotypic profiling problems. We consider several combinations of active learning strategies and popular SML methods. Our results show that active learning significantly reduces the time cost and can be used to reveal the same phenotypic targets identified using SML. We also identify combinations of active learning strategies and SML methods which perform better than others on the phenotypic profiling problems we studied. © 2014 Society for Laboratory Automation and Screening.

Student profiling on university co-curricular activities using cluster analysis

Science.gov (United States)

Rajenthran, Hemabegai A./P.; Shaharanee, Izwan Nizal Mohd; Jamil, Jastini Mohd.

2017-11-01

In higher learning institutions, the co-curricular programs are needed for the graduation besides the standard academic programs. By actively participating in co-curricular, students can attain many of soft skills and proficiencies besides learning and adopting campus environment, community and traditions. Co-curricular activities are implemented by universities mainly for the refinement of the academic achievement along with the social development. This studies aimed to analyse the academic profile of the co-curricular students among uniform units. The main objective of study is to develop a profile of student co-curricular activities in uniform units. Additionally, several variables has been selected to serve as the characteristics for student co-curricular profile. The findings of this study demonstrate the practicality of clustering technique to investigate student's profiles and allow for a better understanding of student's behavior and co-curriculum activities.

Profiling plasma extracellular vesicle by pluronic block-copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion.

Science.gov (United States)

Zhong, Zhenyu; Rosenow, Matthew; Xiao, Nick; Spetzler, David

2018-01-01

Extracellular vesicle (EV)-based liquid biopsies have been proposed to be a readily obtainable biological substrate recently for both profiling and diagnostics purposes. Development of a fast and reliable preparation protocol to enrich such small particles could accelerate the discovery of informative, disease-related biomarkers. Though multiple EV enrichment protocols are available, in terms of efficiency, reproducibility and simplicity, precipitation-based methods are most amenable to studies with large numbers of subjects. However, the selectivity of the precipitation becomes critical. Here, we present a simple plasma EV enrichment protocol based on pluronic block copolymer. The enriched plasma EV was able to be verified by multiple platforms. Our results showed that the particles enriched from plasma by the copolymer were EV size vesicles with membrane structure; proteomic profiling showed that EV-related proteins were significantly enriched, while high-abundant plasma proteins were significantly reduced in comparison to other precipitation-based enrichment methods. Next-generation sequencing confirmed the existence of various RNA species that have been observed in EVs from previous studies. Small RNA sequencing showed enriched species compared to the corresponding plasma. Moreover, plasma EVs enriched from 20 advanced breast cancer patients and 20 age-matched non-cancer controls were profiled by semi-quantitative mass spectrometry. Protein features were further screened by EV proteomic profiles generated from four breast cancer cell lines, and then selected in cross-validation models. A total of 60 protein features that highly contributed in model prediction were identified. Interestingly, a large portion of these features were associated with breast cancer aggression, metastasis as well as invasion, consistent with the advanced clinical stage of the patients. In summary, we have developed a plasma EV enrichment method with improved precipitation selectivity

Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria.

Science.gov (United States)

Kacerovsky, Marian; Celec, Peter; Vlkova, Barbora; Skogstrand, Kristin; Hougaard, David M; Cobo, Teresa; Jacobsson, Bo

2013-01-01

This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

MOLECULAR DOCKING AND DYNAMICS STUDIES ON THE PROTEIN-PROTEIN INTERACTIONS OF ELECTRICALLY ACTIVE PILIN NANOWIRES OF GEOBACTER SULFURREDUCENS.

Directory of Open Access Journals (Sweden)

D. Jeya Sundara Sharmila1 *

2017-06-01

Full Text Available Molecular interactions are key aspects in biological recognitions applicable in nano/micro systems. Bacterial nanowires are pilus filament based structures that can conduct electrons. The transport of electron is proposed to be facilitated by filamentous fibers made up of polymeric assemblies of proteins called pilin. Geobacter sulfurreducens is capable of delivering electrons through extracellular electron transport (EET by employing conductive nanowires, which are pilin proteins composed of type IV subunit PilA. Protein-protein interactions play an important role in the stabilization of the pilin nanowire assembly complex and it contains transmembrane (TM domain. In current study, protein-protein docking and multiple molecular dynamic (MD simulations were performed to understand the binding mode of pilin nanowires. The MD result explains the conformational behavior and folding of pilin nanowires in water environment in different time scale duration 20, 5, 5, 10 and 20ns (total of 60ns. Direct hydrogen bonds and water mediated hydrogen bonds that play a crucial role during the simulation were investigated. The conformational state, folding, end-toend distance profile and hydrogen bonding behavior had indicated that the Geobacter sulfurreducens pilin nanowires have electrical conductivity properties.

TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

Science.gov (United States)

Feller, Georges

2010-08-01

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers.

Science.gov (United States)

Kasari, Marje; Padrik, Peeter; Vaasa, Angela; Saar, Kristi; Leppik, Krista; Soplepmann, Jaan; Uri, Asko

2012-03-15

A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation. Copyright © 2012 Elsevier Inc. All rights reserved.

Mining the active proteome of Arabidopsis thaliana

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Renier A. L. Van Der Hoorn

2011-11-01

Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

Accelerating Information Retrieval from Profile Hidden Markov Model Databases.

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Ahmad Tamimi

Full Text Available Profile Hidden Markov Model (Profile-HMM is an efficient statistical approach to represent protein families. Currently, several databases maintain valuable protein sequence information as profile-HMMs. There is an increasing interest to improve the efficiency of searching Profile-HMM databases to detect sequence-profile or profile-profile homology. However, most efforts to enhance searching efficiency have been focusing on improving the alignment algorithms. Although the performance of these algorithms is fairly acceptable, the growing size of these databases, as well as the increasing demand for using batch query searching approach, are strong motivations that call for further enhancement of information retrieval from profile-HMM databases. This work presents a heuristic method to accelerate the current profile-HMM homology searching approaches. The method works by cluster-based remodeling of the database to reduce the search space, rather than focusing on the alignment algorithms. Using different clustering techniques, 4284 TIGRFAMs profiles were clustered based on their similarities. A representative for each cluster was assigned. To enhance sensitivity, we proposed an extended step that allows overlapping among clusters. A validation benchmark of 6000 randomly selected protein sequences was used to query the clustered profiles. To evaluate the efficiency of our approach, speed and recall values were measured and compared with the sequential search approach. Using hierarchical, k-means, and connected component clustering techniques followed by the extended overlapping step, we obtained an average reduction in time of 41%, and an average recall of 96%. Our results demonstrate that representation of profile-HMMs using a clustering-based approach can significantly accelerate data retrieval from profile-HMM databases.

Proteomic Profiling Comparing the Effects of Different Heat Treatments on Camel (Camelus dromedarius) Milk Whey Proteins.

Science.gov (United States)

Benabdelkamel, Hicham; Masood, Afshan; Alanazi, Ibrahim O; Alzahrani, Dunia A; Alrabiah, Deema K; AlYahya, Sami A; Alfadda, Assim A

2017-03-28

Camel milk is consumed in the Middle East because of its high nutritional value. Traditional heating methods and the duration of heating affect the protein content and nutritional quality of the milk. We examined the denaturation of whey proteins in camel milk by assessing the effects of temperature on the whey protein profile at room temperature (RT), moderate heating at 63 °C, and at 98 °C, for 1 h. The qualitative and quantitative variations in the whey proteins before and after heat treatments were determined using quantitative 2D-difference in gel electrophoresis (DIGE)-mass spectrometry. Qualitative gel image analysis revealed a similar spot distribution between samples at RT and those heated at 63 °C, while the spot distribution between RT and samples heated at 98 °C differed. One hundred sixteen protein spots were determined to be significantly different ( p protein spots were decreased in common in both the heat-treated samples and an additional 25 spots were further decreased in the 98 °C sample. The proteins with decreased abundance included serum albumin, lactadherin, fibrinogen β and γ chain, lactotransferrin, active receptor type-2A, arginase-1, glutathione peroxidase-1 and, thiopurine S, etc. Eight protein spots were increased in common to both the samples when compared to RT and included α-lactalbumin, a glycosylation-dependent cell adhesion molecule. Whey proteins present in camel milk were less affected by heating at 63 °C than at 98 °C. This experimental study showed that denaturation increased significantly as the temperature increased from 63 to 98 °C.

ELEVATED CHOLESTERYL ESTER TRANSFER PROTEIN-ACTIVITY IN IDDM MEN WHO SMOKE - POSSIBLE FACTOR FOR UNFAVORABLE LIPOPROTEIN PROFILE

NARCIS (Netherlands)

DULLAART, RPF; GROENER, JEM; DIKKESCHEI, BD; ERKELENS, DW; DOORENBOS, H

Objectives: To determine the effect of cigarette smoking on the activity of cholesteryl ester transfer protein (CETP) and high-density (HDL), low-density (LDL), and very-low-density (VLDL) lipoproteins in insulin-dependent diabetic (IDDM) men with microvascular complications. Research Design and

Expression Profiling of Mitogen-Activated Protein Kinase Genes Reveals Their Evolutionary and Functional Diversity in Different Rubber Tree (Hevea brasiliensis Cultivars

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Xiang Jin

2017-10-01

Full Text Available Rubber tree (Hevea brasiliensis is the only commercially cultivated plant for producing natural rubber, one of the most essential industrial raw materials. Knowledge of the evolutionary and functional characteristics of kinases in H. brasiliensis is limited because of the long growth period and lack of well annotated genome information. Here, we reported mitogen-activated protein kinases in H. brasiliensis (HbMPKs by manually checking and correcting the rubber tree genome. Of the 20 identified HbMPKs, four members were validated by proteomic data. Protein motif and phylogenetic analyses classified these members into four known groups comprising Thr-Glu-Tyr (TEY and Thr-Asp-Tyr (TDY domains, respectively. Evolutionary and syntenic analyses suggested four duplication events: HbMPK3/HbMPK6, HbMPK8/HbMPK9/HbMPK15, HbMPK10/HbMPK12 and HbMPK11/HbMPK16/HbMPK19. Expression profiling of the identified HbMPKs in roots, stems, leaves and latex obtained from three cultivars with different latex yield ability revealed tissue- and variety-expression specificity of HbMPK paralogues. Gene expression patterns under osmotic, oxidative, salt and cold stresses, combined with cis-element distribution analyses, indicated different regulation patterns of HbMPK paralogues. Further, Ka/Ks and Tajima analyses suggested an accelerated evolutionary rate in paralogues HbMPK10/12. These results revealed HbMPKs have diverse functions in natural rubber biosynthesis, and highlighted the potential possibility of using MPKs to improve stress tolerance in future rubber tree breeding.

Mitogen-activated protein kinases mediate Mycobacterium ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

Science.gov (United States)

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

A novel protease activity assay using a protease-responsive chaperone protein

International Nuclear Information System (INIS)

Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

2009-01-01

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

A novel protease activity assay using a protease-responsive chaperone protein

Energy Technology Data Exchange (ETDEWEB)

Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

2009-06-05

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

Science.gov (United States)

Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

2013-05-17

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

Science.gov (United States)

Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

2018-01-24

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Real Mission Profile Based Lifetime Estimation of Fuel-cell Power Converter

DEFF Research Database (Denmark)

Zhou, Dao; Wang, Huai; Blaabjerg, Frede

2016-01-01

. This paper describes a lifetime prediction method for the power semiconductors used in the power conditioning of a fuel cell based backup system, considering both the long-term standby mode and active operation mode. The annual ambient temperature profile is taken into account to estimate its impact...... on the degradation of MOSFETs during the standby mode. At the presence of power outages, the backup system is activated into the operation mode and the MOSFETs withstand additional thermal stresses due to power losses. A study case of a 1 kW backup system is presented with two annual mission profiles in Denmark...... and India, respectively. The ambient temperature, occurrence frequency of power outages, active operation time and power levels are considered for the lifetime prediction of the applied MOSFETs. Comparisons of the accumulated lifetime consumptions are performed between standby mode and operation mode...

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

International Nuclear Information System (INIS)

Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

2008-01-01

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

Science.gov (United States)

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

Science.gov (United States)

Zeke, András; Misheva, Mariya

2016-01-01

SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

Postprandial lipemia detects the effect of soy protein on cardiovascular disease risk compared with the fasting lipid profile.

Science.gov (United States)

Santo, Antonio S; Santo, Ariana M; Browne, Richard W; Burton, Harold; Leddy, John J; Horvath, Steven M; Horvath, Peter J

2010-12-01

Studies examining the effect of soy protein on cardiovascular disease (CVD) risk factors have not taken advantage of the postprandial state as an adjunct to the fasting lipid profile. The American Heart Association has acknowledged the efficacy of soy protein in reducing CVD risk factors to be limited. We hypothesized that the postprandial state would be more sensitive to any favorable changes associated with consuming soy protein compared with the fasting lipid profile. Furthermore, the presence of isoflavones in soy would enhance this effect. Thirty sedentary males aged 18-30 years were randomly assigned to milk protein (Milk), isoflavone-poor soy (Soy-), or isoflavone-rich soy (Soy+). Usual diets were supplemented with 25 g/day of protein for 28 days. Serum samples were collected before and after supplementation in a fasted state and postprandially at 30, 60, 120, 240, and 360 min after a high-fat, 1,000 kcal shake. Triacylglycerol (TAG), total cholesterol, non-esterified fatty acids, apolipoproteins B-100 and A-I and glucose concentrations were quantified. Fasting concentrations were not different after any protein supplementation. Postprandial TAG and TAG AUC increased after Soy-consumption supporting the postprandial state as a more sensitive indicator of soy ingestion effects on CVD risk factors compared with the fasting lipid profile. Furthermore, the absence of isoflavones in soy protein may have deleterious consequences on purported cardio-protective effects.

Protein Hydrolysis from Catfish Prepared by Papain Enzyme and Antioxidant Activity of Hydrolyzate

Directory of Open Access Journals (Sweden)

Ace Baehaki

2015-12-01

Full Text Available The objective of this research was to make a protein hydrolysates from catfish (Pangasius pangasius enzymatically using papain enzyme and analyzed the antioxidant activity of protein hydrolysates produced. The research used the method completely randomized design with two replications the treatment were the difference concentration of the papain enzyme (0%, 1%, 2%, 3%, 4%, 5%, and 6%. The parameters of research were antioxidative activity using DPPH (2,2-difenil-1–pikrilhidrazil, protein content, and molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. The results showed that catfish protein hydrolysates prepared by papain enzyme has antioxidative activity. The highest degree of hydrolysis was 71.98% at enzyme concentration of 6%. Based on the DPPH scavenging method catfish protein hydrolysates has the antioxidative activity with the value 37.85-67.62%. The protein content of catfish protein hydrolysates were 20.86-54.47 mg/ml. The molecular weight of catfish protein hydrolyzates were 11.90-65.20 kDa.

Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

Science.gov (United States)

Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

2006-09-06

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

Science.gov (United States)

Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

2016-03-01

T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

Active epilepsy prevalence, the treatment gap, and treatment gap risk profile in eastern China: A population-based study.

Science.gov (United States)

Ding, Xiaoyan; Zheng, Yang; Guo, Yi; Shen, Chunhong; Wang, Shan; Chen, Feng; Yan, Shengqiang; Ding, Meiping

2018-01-01

We measured the prevalence of active epilepsy and investigated the treatment gap and treatment gap risk profile in eastern China. This was a cross-sectional population-based survey conducted in Zhejiang, China, from October 2013 to March 2014. A total 54,976 people were selected using multi-stage cluster sampling. A two-stage questionnaire-based process was used to identify patients with active epilepsy and to record their demographic, socioeconomic, and epilepsy-related features. Logistic regression analysis was used to analyze risk factors of the treatment gap in eastern China, as adjusted for age and sex. We interviewed 50,035 people; 118 had active epilepsy (2.4‰), among which the treatment gap was 58.5%. In multivariate analysis, failure to receive appropriate antiepileptic treatment was associated with higher seizure frequency of 12-23 times per year (adjusted odds ratio=6.874; 95% confidence interval [CI]=2.372-19.918), >24 times per year (adjusted odds ratio=19.623; 95% CI=4.999-77.024), and a lack of health insurance (adjusted odds ratio=7.284; 95% CI=1.321-40.154). Eastern China has relatively lower prevalence of active epilepsy and smaller treatment gap. Interventions aimed at reducing seizure frequency, improving the health insurance system should be investigated as potential targets to further bridge the treatment gap. Copyright © 2017 Elsevier Inc. All rights reserved.

Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria.

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Marian Kacerovsky

Full Text Available OBJECTIVE: This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. METHODS: A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. RESULTS: The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. CONCLUSIONS: The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

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

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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.

Activity-based protein profiling of glucosidases, fucosidases and glucuronidases

NARCIS (Netherlands)

Jiang, J.

2016-01-01

Glycoside hydrolases (GHs), enzymes that catalyze the hydrolytic cleavage of glycosidic bonds, receive continuing interest both in fundamental and applied biology and biomedicine. Lysosomal storage disorders (LSDs) are caused by inborn metabolic errors due to deficiency in specific lysosomal

Nε-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure-Activity Relationships, and Pharmacokinetic Profiling.

Science.gov (United States)

Wodtke, Robert; Hauser, Christoph; Ruiz-Gómez, Gloria; Jäckel, Elisabeth; Bauer, David; Lohse, Martin; Wong, Alan; Pufe, Johanna; Ludwig, Friedrich-Alexander; Fischer, Steffen; Hauser, Sandra; Greif, Dieter; Pisabarro, M Teresa; Pietzsch, Jens; Pietsch, Markus; Löser, Reik

2018-05-24

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N ε -acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M -1 s -1 , which resulted in comprehensive structure-activity relationships. Structure-activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the molecular recognition for this inhibitor class within the active site of TGase 2. Selectivity profiling of selected compounds for other transglutaminases demonstrated an excellent selectivity toward transglutaminase 2. Furthermore, an initial pharmacokinetic profiling of selected inhibitors was performed, including the assessment of potential membrane permeability and liver microsomal stability.

Analysis on Protein Profile and Amino Acid of Edible Bird's Nest (Collocalia Fuchiphaga) From Painan

OpenAIRE

Elfita, Lina

2014-01-01

This study was aimed to analyze protein profile and amino acid composition of bird nest from Painan, Pesisir Selatan Distric, West Sumatra. Protein analysis was performed by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE), meanwhile High Performance Liquid Chromatography (HPLC) was used for analysis of amino acid. Analysis on water extract of bird nest by SDS-PAGE showed six bands which correspond to molecular protein which had molecular weight of 147.2; 142.6; 133.4; 73...

RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3

International Nuclear Information System (INIS)

Miles, Godfrey P.; Samuel, Marcus A.; Zhang Yuelin; Ellis, Brian E.

2005-01-01

The recent increase in tropospheric ozone (O 3 ) concentrations promotes additional oxidative stress through the production of reactive oxygen species (ROS) in plant tissues, resulting in the activation of genes whose products enable the stressed cells to retain their integrity and function. This response is made possible by an integration of highly regulated signaling networks that mediate the perception of, and response to, this oxidative assault. In Arabidopsis thaliana, ROS-induced signaling has been shown to flow through a protein phosphorylation cascade involving the mitogen-activated protein kinases (MAPKs) AtMPK3 (MPK3) and AtMPK6 (MPK6). We found that RNAi-mediated silencing of MPK6 renders the plant more sensitive to ozone, as determined by visible leaf damage. The MPK6-RNAi genotype also displayed a more intense and prolonged activation of MPK3 compared to that of WT plants. An MPK3 loss-of-function genotype is similarly very sensitive to ozone, and displays an abnormally prolonged MPK6 activation profile, suggesting reciprocity in regulation between these two MAPKs. - MPK6 is pivotal in the overall response to oxidative stress and regulation of MPK3 in Arabidopsis thaliana

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

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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

Protein profiling in serum after traumatic brain injury in rats reveals potential injury markers.

Science.gov (United States)

Thelin, Eric Peter; Just, David; Frostell, Arvid; Häggmark-Månberg, Anna; Risling, Mårten; Svensson, Mikael; Nilsson, Peter; Bellander, Bo-Michael

2018-03-15

The serum proteome following traumatic brain injury (TBI) could provide information for outcome prediction and injury monitoring. The aim with this affinity proteomic study was to identify serum proteins over time and between normoxic and hypoxic conditions in focal TBI. Sprague Dawley rats (n=73) received a 3mm deep controlled cortical impact ("severe injury"). Following injury, the rats inhaled either a normoxic (22% O 2 ) or hypoxic (11% O 2 ) air mixture for 30min before resuscitation. The rats were sacrificed at day 1, 3, 7, 14 and 28 after trauma. A total of 204 antibodies targeting 143 unique proteins of interest in TBI research, were selected. The sample proteome was analyzed in a suspension bead array set-up. Comparative statistics and factor analysis were used to detect differences as well as variance in the data. We found that complement factor 9 (C9), complement factor B (CFB) and aldolase c (ALDOC) were detected at higher levels the first days after trauma. In contrast, hypoxia inducing factor (HIF)1α, amyloid precursor protein (APP) and WBSCR17 increased over the subsequent weeks. S100A9 levels were higher in hypoxic-compared to normoxic rats, together with a majority of the analyzed proteins, albeit few reached statistical significance. The principal component analysis revealed a variance in the data, highlighting clusters of proteins. Protein profiling of serum following TBI using an antibody based microarray revealed temporal changes of several proteins over an extended period of up to four weeks. Further studies are warranted to confirm our findings. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Evaluation of a community-based participatory physical activity promotion project: effect on cardiovascular disease risk profiles of school employees

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Kobza Cee E

2010-06-01

Full Text Available Abstract Background The efficacy of physical activity in improving cardiovascular disease (CVD risk profiles has been well established. However, the effectiveness of health promotion programs implemented at the community level remains controversial. This study evaluated a school-based work-site physical activity program. Methods Using a community-based participatory research model, a work-site wellness intervention was implemented in a rural public school system in Southwestern Oklahoma. During the 2005-2006 school year, 187 participants (mean age 45 years completed a pre intervention screening for CVD risk factors followed by a physical activity promotion program. Post intervention screening was conducted after a 6 month period. During both screening sessions, body composition, blood pressure, lipids, glucose and self-reported physical activity levels were assessed. The focus of the intervention was on promoting physical activity. Opportunities for in school physical activity were created by marking hallways, adding a treadmill in each school, and allowing teachers to use planning periods for physical activity. Results During the post intervention screening, compared to pre intervention levels, participants had lower total, low, and high density lipoprotein-cholesterol (t = 5.9, p Conclusions A successful participatory program was associated with improvements in several CVD risk factors among school employees. Limitations of this study such as seasonal variation in the outcome variables and lack of a control group limit our ability to draw solid conclusions about the effectiveness of the intervention.

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

International Nuclear Information System (INIS)

Yoon, Joon Kee; Lee, Myung Hoon; Joh, Chul Woo; Yoon, Seok Nam; Soh, Eui Young

2003-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Yoon, Joon Kee; Lee, Myung Hoon; Joh, Chul Woo; Yoon, Seok Nam; Soh, Eui Young [College of Medicine, Univ. of Ajou, Suwon (Korea, Republic of)

2003-07-01

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.

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

A protein relational database and protein family knowledge bases to facilitate structure-based design analyses.

Science.gov (United States)

Mobilio, Dominick; Walker, Gary; Brooijmans, Natasja; Nilakantan, Ramaswamy; Denny, R Aldrin; Dejoannis, Jason; Feyfant, Eric; Kowticwar, Rupesh K; Mankala, Jyoti; Palli, Satish; Punyamantula, Sairam; Tatipally, Maneesh; John, Reji K; Humblet, Christine

2010-08-01

The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom-atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid-centroid and centroid-atom distances and angles have also been included permitting queries for pi-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.

Developing Novel Protein-based Materials using Ultrabithorax: Production, Characterization, and Functionalization

Science.gov (United States)

Huang, Zhao

2011-12-01

Compared to 'conventional' materials made from metal, glass, or ceramics, protein-based materials have unique mechanical properties. Furthermore, the morphology, mechanical properties, and functionality of protein-based materials may be optimized via sequence engineering for use in a variety of applications, including textile materials, biosensors, and tissue engineering scaffolds. The development of recombinant DNA technology has enabled the production and engineering of protein-based materials ex vivo. However, harsh production conditions can compromise the mechanical properties of protein-based materials and diminish their ability to incorporate functional proteins. Developing a new generation of protein-based materials is crucial to (i) improve materials assembly conditions, (ii) create novel mechanical properties, and (iii) expand the capacity to carry functional protein/peptide sequences. This thesis describes development of novel protein-based materials using Ultrabithorax, a member of the Hox family of proteins that regulate developmental pathways in Drosophila melanogaster. The experiments presented (i) establish the conditions required for the assembly of Ubx-based materials, (ii) generate a wide range of Ubx morphologies, (iii) examine the mechanical properties of Ubx fibers, (iv) incorporate protein functions to Ubx-based materials via gene fusion, (v) pattern protein functions within the Ubx materials, and (vi) examine the biocompatibility of Ubx materials in vitro. Ubx-based materials assemble at mild conditions compatible with protein folding and activity, which enables Ubx chimeric materials to retain the function of appended proteins in spatial patterns determined by materials assembly. Ubx-based materials also display mechanical properties comparable to existing protein-based materials and demonstrate good biocompatibility with living cells in vitro. Taken together, this research demonstrates the unique features and future potential of novel Ubx-based

Chemical characteristics and enhanced hepatoprotective activities of Maillard reaction products derived from milk protein-sugar system.

Science.gov (United States)

Oh, Nam Su; Young Lee, Ji; Lee, Hyun Ah; Joung, Jae Yeon; Shin, Yong Kook; Kim, Sae Hun; Kim, Younghoon; Lee, Kwang Won

2016-02-01

The objective of this study was to investigate the characteristics, antioxidative properties, and hepatoprotective effects of Maillard reaction products (MRP) from milk protein reacted with sugars. The MRP were obtained from milk protein, whey protein concentrates and sodium caseinate, using 2 types of sugars, lactose and glucose, by heating the mixture at 55°C for 7d in a sodium phosphate buffer (pH 7.4). Changes in the chemical modification of the milk protein were monitored by measuring the protein-bound carbonyls and PAGE protein profiles. The results showed that the amount of protein-bound carbonyls increased after Maillard reaction (MR). In addition, sodium dodecyl sulfate-PAGE analysis indicated a formation of high-molecular weight complexes through MR. The modification sites induced by MR of milk protein were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of tryptic-digested gel spots of MRP. As a result, modification and their localization in AA sequence of MRP was identified. Also, the MRP showed higher antioxidant activities than the intact milk protein, and they reduced intracellular reactive oxygen species production and inhibited the depletion of the reduced glutathione concentrations in the HepG2 cells. In particular, glucose-sodium caseinate MRP showed the highest biological activities among all MRP. Therefore, these results suggest that the MRP from milk protein reacting with sugars possess effective antioxidant activity and have a protective ability against oxidative damage. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Variation in the protein profiles in the gamma-irradiated chick pea (Cicer arietinum L.) seeds

International Nuclear Information System (INIS)

Farook, S.A.F.; Nizam, Jafar

1978-01-01

Water soluble seed proteins from the control as well as gamma ray treated material from the M 2 generation of Chick pea (Cicer arietinum L.) were separated by disc electrophoresis using 7.5 percent poly acrylamide gels. Average Rf values and percentage of similarity values were calculated. The comparisons of number and Rf values of protein bands were made to elucidate the differences in the treated material. Differences obtained in the seed protein profiles of the treated material suggest the presence of the qualitative variation in the proteins. Attempts were made to correlate the variation in the protein bands with the morphological changes in the mutants. (author)

Urban Adolescents' Out-of-School Activity Profiles: Associations with Youth, Family, and School Transition Characteristics

Science.gov (United States)

Pedersen, Sara

2005-01-01

This study applied individual growth trajectory analyses and person-oriented analysis to identify common profiles of out-of-school activity engagement trajectories among racially and ethnically diverse inner city teens (N = 1,430). On average, teens exhibited declining trajectories of participation in school-based and team sports activities and…

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

Science.gov (United States)

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

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

Directory of Open Access Journals (Sweden)

A. A. Grazhdantseva

2010-01-01

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

Physical activity and sleep profiles in Finnish men and women.

Science.gov (United States)

Wennman, Heini; Kronholm, Erkki; Partonen, Timo; Tolvanen, Asko; Peltonen, Markku; Vasankari, Tommi; Borodulin, Katja

2014-01-27

Physical activity (PA) and sleep are related to cardiovascular diseases (CVD) and their risk factors. The interrelationship between these behaviors has been studied, but there remain questions regarding the association of different types of PA, such as occupational, commuting, and leisure time to sleep, including quality, duration and sufficiency. It is also unclear to what extent sleep affects peoples' PA levels and patterns. Our aim is to investigate the interrelationship between PA and sleep behaviors in the Finnish population, including employment status and gender. The study comprised population based data from the FINRISK 2012 Study. A stratified, random sample of 10,000 Finns, 25 to 74 years-old, were sent a questionnaire and an invitation to a health examination. The participation rate was 64% (n = 6,414). Latent class analysis was used to search for different underlying profiles of PA and sleep behavior in men and women, respectively. Models with one through five latent profiles were fitted to the data. Based on fit indicators, a four-class model for men and women, respectively, was decided to be the best fitted model. Four different profiles of PA and sleep were found in both men and women. The most common profile of men comprised 45% of the total participants, and in women, 47%. These profiles were distinguished by probabilities for high leisure time PA and sleep, subjectively rated as sufficient, as well as sleep duration of 7-7.9 hours. The least common profiles represented 5% (men) and 11% (women) of the population, and were characterized by probabilities for physical inactivity, short sleep, and evening type for women and morning type for men. There was also one profile in both genders characterized by likelihood for both high occupational PA and subjectively experienced insufficient sleep. The use of latent class analysis in investigating the interrelationship between PA and sleep is a novel perspective. The method provides information on the

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

Science.gov (United States)

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

2017-10-01

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antioxidant activity and protein-polyphenol interactions in a pomegranate (Punica granatum L.) yogurt.

Science.gov (United States)

Trigueros, Lorena; Wojdyło, Aneta; Sendra, Esther

2014-07-09

Pomegranate juice (PGJ) is rich in phenolics which are potent antioxidants but also prone to interact with proteins. A yogurt rich in PGJ (40%) made from arils was elaborated (PGY) to determine the antioxidant activity and to estimate the phenolics-proteins interaction during 28 days of cold storage. Juice, yogurts, and protein-free permeates were analyzed for phenolic composition. Yogurt fermentation modified the anthocyanin profile of the initial PGJ, especially the content in cyanidin-3-O-glucoside. During storage, individual anthocyanin content in PGY decreased but it did not modify yogurt color. The analysis of permeates revealed that the degree of phenol-protein interaction depends on the type of phenolic, ellagic acid and dephinidin-3,5-O-diglucoside being the least bound phenolic compounds. The presence of PGJ in yogurt enhanced radical scavenging performance, whereas all the observed ferric reducing power ability of PGY was strictly due to the PGJ present. The 84.73% of total anthocyanins remained bound to proteins at the first day of storage and 90.06% after 28 days of cold storage, revealing the high affinity of anthocyanins for milk proteins.

Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

Directory of Open Access Journals (Sweden)

Mohammed Akli eAyoub

2013-12-01

Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.

Assessment of leisure-time physical activity for the prediction of inflammatory status and cardiometabolic profile.

Science.gov (United States)

Pires, Milena Monfort; Salvador, Emanuel P; Siqueira-Catania, Antonela; Folchetti, Luciana D; Cezaretto, Adriana; Ferreira, Sandra Roberta G

2012-11-01

Associations of leisure-time physical activity (LTPA), commuting and total physical activity with inflammatory markers, insulin resistance and metabolic profile in individuals at high cardiometabolic risk were investigated. This was a cross-sectional study. A total of 193 prediabetic adults were compared according to physical activity levels measured by the international physical activity questionnaire; p for trend and logistic regression was employed. The most active subset showed lower BMI and abdominal circumference, reaching significance only for LTPA (p for trend=0.02). Lipid profile improved with increased physical activity levels. Interleukin-6 decreased with increased total physical activity and LTPA (p for trend=0.02 and 0.03, respectively), while adiponectin increased in more active subsets for LTPA (p for trend=0.03). Elevation in adjusted OR for hypercholesterolemia was significant for lower LTPA durations (p for trend=0.04). High apolipoprotein B/apolipoprotein A ratio was inversely associated with LTPA, commuting and total physical activity. Increase in adjusted OR for insulin resistance was found from the highest to the lowest category of LTPA (p for trend=0.04) but significance disappeared after adjustments for BMI and energy intake. No association of increased C-reactive protein with physical activity domains was observed. In general, the associations of LTPA, but not commuting or total physical activity, with markers of cardiometabolic risk reinforces the importance of initiatives to increase this domain in programs for the prevention of lifestyle-related diseases. Copyright © 2012 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

A new intelligent algorithm to create a profile for user based on web interactions

Directory of Open Access Journals (Sweden)

Zeinab khademali

2013-04-01

Full Text Available This paper presents a method to classify the web user’s navigation patterns automatically. The proposed model of this paper classifies user’s navigation patterns and predicts his/her upcoming requirements. To create users’ profile, a new method is introduced by recording user’s settings active and user’s similarity measurement with neighboring users. The proposed model is capable of creating the profile implicitly. Besides, it updates the profile based on created changes. In fact, we try to improve the function of recommender engine using user’s navigation patterns and clustering. The method is based on user’s navigation patterns and is able to present the result of recommender engine based on user’s requirement and interest. In addition, this method has the ability to help customize websites, more efficiently.

A procedure to analyze surface profiles of the protein molecules visualized by quick-freeze deep-etch replica electron microscopy

Energy Technology Data Exchange (ETDEWEB)

Kimori, Yoshitaka [Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502 (Japan); Oguchi, Yosuke [Department of Electric Engineering, Kogakuin University, Hachioji, Tokyo 192-0015 (Japan); Ichise, Norihiko [Department of Visual Communication, Komazawa Women' s University, Inagi, Tokyo 206-8511 (Japan); Baba, Norio [Department of Electric Engineering, Kogakuin University, Hachioji, Tokyo 192-0015 (Japan); Katayama, Eisaku [Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)]. E-mail: ekatayam@ims.u-tokyo.ac.jp

2007-01-15

Quick-freeze deep-etch replica electron microscopy gives high contrast snapshots of individual protein molecules under physiological conditions in vitro or in situ. The images show delicate internal pattern, possibly reflecting the rotary-shadowed surface profile of the molecule. As a step to build the new system for the 'Structural analysis of single molecules', we propose a procedure to quantitatively characterize the structural property of individual molecules; e.g. conformational type and precise view-angle of the molecules, if the crystallographic structure of the target molecule is available. This paper presents a framework to determine the observed face of the protein molecule by analyzing the surface profile of individual molecules visualized in freeze-replica specimens. A comprehensive set of rotary-shadowed views of the protein molecule was artificially generated from the available atomic coordinates using light-rendering software. Exploiting new mathematical morphology-based image filter, characteristic features were extracted from each image and stored as template. Similar features were extracted from the true replica image and the most likely projection angle and the conformation of the observed particle were determined by quantitative comparison with a set of archived images. The performance and the robustness of the procedure were examined with myosin head structure in defined configuration for actual application.

A procedure to analyze surface profiles of the protein molecules visualized by quick-freeze deep-etch replica electron microscopy

International Nuclear Information System (INIS)

Kimori, Yoshitaka; Oguchi, Yosuke; Ichise, Norihiko; Baba, Norio; Katayama, Eisaku

2007-01-01

Quick-freeze deep-etch replica electron microscopy gives high contrast snapshots of individual protein molecules under physiological conditions in vitro or in situ. The images show delicate internal pattern, possibly reflecting the rotary-shadowed surface profile of the molecule. As a step to build the new system for the 'Structural analysis of single molecules', we propose a procedure to quantitatively characterize the structural property of individual molecules; e.g. conformational type and precise view-angle of the molecules, if the crystallographic structure of the target molecule is available. This paper presents a framework to determine the observed face of the protein molecule by analyzing the surface profile of individual molecules visualized in freeze-replica specimens. A comprehensive set of rotary-shadowed views of the protein molecule was artificially generated from the available atomic coordinates using light-rendering software. Exploiting new mathematical morphology-based image filter, characteristic features were extracted from each image and stored as template. Similar features were extracted from the true replica image and the most likely projection angle and the conformation of the observed particle were determined by quantitative comparison with a set of archived images. The performance and the robustness of the procedure were examined with myosin head structure in defined configuration for actual application

Concentration profiles near an activated enzyme.

Science.gov (United States)

Park, Soohyung; Agmon, Noam

2008-09-25

When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

A Physiologically Based Pharmacokinetic Model to Predict the Pharmacokinetics of Highly Protein-Bound Drugs and Impact of Errors in Plasma Protein Binding

Science.gov (United States)

Ye, Min; Nagar, Swati; Korzekwa, Ken

2015-01-01

Predicting the pharmacokinetics of highly protein-bound drugs is difficult. Also, since historical plasma protein binding data was often collected using unbuffered plasma, the resulting inaccurate binding data could contribute to incorrect predictions. This study uses a generic physiologically based pharmacokinetic (PBPK) model to predict human plasma concentration-time profiles for 22 highly protein-bound drugs. Tissue distribution was estimated from in vitro drug lipophilicity data, plasma protein binding, and blood: plasma ratio. Clearance was predicted with a well-stirred liver model. Underestimated hepatic clearance for acidic and neutral compounds was corrected by an empirical scaling factor. Predicted values (pharmacokinetic parameters, plasma concentration-time profile) were compared with observed data to evaluate model accuracy. Of the 22 drugs, less than a 2-fold error was obtained for terminal elimination half-life (t1/2, 100% of drugs), peak plasma concentration (Cmax, 100%), area under the plasma concentration-time curve (AUC0–t, 95.4%), clearance (CLh, 95.4%), mean retention time (MRT, 95.4%), and steady state volume (Vss, 90.9%). The impact of fup errors on CLh and Vss prediction was evaluated. Errors in fup resulted in proportional errors in clearance prediction for low-clearance compounds, and in Vss prediction for high-volume neutral drugs. For high-volume basic drugs, errors in fup did not propagate to errors in Vss prediction. This is due to the cancellation of errors in the calculations for tissue partitioning of basic drugs. Overall, plasma profiles were well simulated with the present PBPK model. PMID:26531057

Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

Science.gov (United States)

Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

2016-02-25

Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2012-02-01

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2009-02-27

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

Science.gov (United States)

Bae, Young-An; Kim, Jeong-Geun; Kong, Yoon

2016-01-01

Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system. Copyright © 2016 Elsevier B.V. All rights reserved.

Alterations in the fatty acid profile, antioxidant enzymes and protein pattern of Biomphalaria alexandrina snails exposed to the pesticides diazinon and profenfos.

Science.gov (United States)

Bakry, Fayez A; El-Hommossany, Karem; Abd El-Atti, Mahmoud; Ismail, Somaya M

2016-04-01

The use of pesticides is widespread in agricultural activities. These pesticides may contaminate the irrigation and drainage systems during agriculture activities and pests' control and then negatively affect the biotic and a biotic component of the polluted water courses. The present study aimed to evaluate the effect of the pesticides diazinon and profenfos on some biological activities of Biomphalaria alexandrina snails such as fatty acid profile, some antioxidant enzymes (thioredoxin reductase (TrxR), sorbitol dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT) as well as glutathione reductase (GR) and lipid peroxidation (LP)) and protein patterns in snails' tissues exposed for 4 weeks to LC10 of diazinon and profenfos. The results showed that the two pesticides caused considerable reduction in survival rates and egg production of treated snails. Identification of fatty acid composition in snail tissues treated with diazinon and profenfos pesticides was carried out using gas-liquid chromatography (GLC). The results declared alteration in fatty acid profile, fluctuation in percentage of long chain and short chain fatty acid contributions either saturated or unsaturated ones, and a decrease in total lipid content in tissues of snails treated with these pesticides. The data demonstrate that there was a significant inhibition in the activities of tissues SOD, CAT, glutathione reductase (GR), TrxR, and SDH in tissues of treated snails, while a significant elevation was detected in LP as compared to the normal control. On the other hand, the electrophoretic pattern of total protein showed differences in number and molecular weights of protein bands due to the treatment of snails. It was concluded that the residues of diazinon and profenfos pesticides in aquatic environments have toxic effects onB. alexandrina snails. © The Author(s) 2013.

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

Directory of Open Access Journals (Sweden)

Zarei Saeed

2011-08-01

Full Text Available Abstract Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula.

Science.gov (United States)

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling; Wang, Yun

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components.

Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

Science.gov (United States)

Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

2015-01-01

Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dietary live yeast alters metabolic profiles, protein biosynthesis and thermal stress tolerance of Drosophila melanogaster.

Science.gov (United States)

Colinet, Hervé; Renault, David

2014-04-01

The impact of nutritional factors on insect's life-history traits such as reproduction and lifespan has been excessively examined; however, nutritional determinant of insect's thermal tolerance has not received a lot of attention. Dietary live yeast represents a prominent source of proteins and amino acids for laboratory-reared drosophilids. In this study, Drosophila melanogaster adults were fed on diets supplemented or not with live yeast. We hypothesized that manipulating nutritional conditions through live yeast supplementation would translate into altered physiology and stress tolerance. We verified how live yeast supplementation affected body mass characteristics, total lipids and proteins, metabolic profiles and cold tolerance (acute and chronic stress). Females fed with live yeast had increased body mass and contained more lipids and proteins. Using GC/MS profiling, we found distinct metabolic fingerprints according to nutritional conditions. Metabolite pathway enrichment analysis corroborated that live yeast supplementation was associated with amino acid and protein biosyntheses. The cold assays revealed that the presence of dietary live yeast greatly promoted cold tolerance. Hence, this study conclusively demonstrates a significant interaction between nutritional conditions and thermal tolerance. Copyright © 2014 Elsevier Inc. All rights reserved.

Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment

Directory of Open Access Journals (Sweden)

Pek Siew Lim

2016-03-01

Full Text Available T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA and calcium ionomycin (I as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278. Keywords: EL4 T cell, Microarray, T cell activation, Inducible genes

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

KAUST Repository

Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

2014-01-01

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

KAUST Repository

Zourelidou, Melina

2014-06-19

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

International Nuclear Information System (INIS)

Yu, P.

2007-01-01

The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

Science.gov (United States)

Djordjevic, Michael A; Chen, Han Cai; Natera, Siria; Van Noorden, Giel; Menzel, Christian; Taylor, Scott; Renard, Clotilde; Geiger, Otto; Weiller, Georg F

2003-06-01

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.