Mirror profile optimization for nano-focusing KB mirror

International Nuclear Information System (INIS)

Zhang Lin; Baker, Robert; Barrett, Ray; Cloetens, Peter; Dabin, Yves

2010-01-01

A KB focusing mirror width profile has been optimized to achieve nano-focusing for the nano-imaging end-station ID22NI at the ESRF. The complete mirror and flexure bender assembly has been modeled in 3D with finite element analysis using ANSYS. Bender stiffness, anticlastic effects and geometrical non-linear effects have been considered. Various points have been studied: anisotropy and crystal orientation, stress in the mirror and bender, actuator resolution and the mirror-bender adhesive bonding... Extremely high performance of the mirror is expected with residual slope error smaller than 0.6 μrad, peak-to-valley, compared to the bent slope of 3000 μrad.

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

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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

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....

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.

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....

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.

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

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

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

Bioorthogonal chemistry: applications in activity-based protein profiling.

Science.gov (United States)

Willems, Lianne I; van der Linden, Wouter A; Li, Nan; Li, Kah-Yee; Liu, Nora; Hoogendoorn, Sascha; van der Marel, Gijs A; Florea, Bogdan I; Overkleeft, Herman S

2011-09-20

of chemical biology research include contributions from many areas of the multifaceted discipline of chemistry, and particularly from organic chemistry. Researchers apply knowledge inherent to organic chemistry, such as reactivity and selectivity, to the manipulation of specific biomolecules in biological samples (cell extracts, living cells, and sometimes even animal models) to gain insight into the biological phenomena in which these molecules participate. In this Account, we highlight some of the recent developments in chemical biology research driven by organic chemistry, with a focus on bioorthogonal chemistry in relation to activity-based protein profiling. The rigorous demands of bioorthogonality have not yet been realized in a truly bioorthogonal reagent pair, but remarkable progress has afforded a range of tangible contributions to chemical biology research. Activity-based protein profiling, which aims to obtain information on the workings of a protein (or protein family) within the larger context of the full biological system, has in particular benefited from these advances. Both activity-based protein profiling and bioorthogonal chemistry have been around for approximately 15 years, and about 8 years ago the two fields very profitably intersected. We expect that each discipline, both separately and in concert, will continue to make important contributions to chemical biology research. © 2011 American Chemical Society

HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features.

Science.gov (United States)

Zaman, Rianon; Chowdhury, Shahana Yasmin; Rashid, Mahmood A; Sharma, Alok; Dehzangi, Abdollah; Shatabda, Swakkhar

2017-01-01

DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM) as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features

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Rianon Zaman

2017-01-01

Full Text Available DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

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.

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...

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.

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...

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.

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...

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.

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

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Lin Wei Jong

2015-05-01

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

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.

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.

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 ...

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

Science.gov (United States)

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

2015-06-30

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

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

Relativistic self-focusing of intense laser beam in thermal collisionless quantum plasma with ramped density profile

Directory of Open Access Journals (Sweden)

S. Zare

2015-04-01

Full Text Available Propagation of a Gaussian x-ray laser beam has been analyzed in collisionless thermal quantum plasma with considering a ramped density profile. In this density profile due to the increase in the plasma density, an earlier and stronger self-focusing effect is noticed where the beam width oscillates with higher frequency and less amplitude. Moreover, the effect of the density profile slope and the initial plasma density on the laser propagation has been studied. It is found that, by increasing the initial density and the ramp slope, the laser beam focuses faster with less oscillation amplitude, smaller laser spot size and more oscillations. Furthermore, a comparison is made among the laser self-focusing in thermal quantum plasma, cold quantum plasma and classical plasma. It is realized that the laser self-focusing in the quantum plasma becomes stronger in comparison with the classical regime.

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 ...

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 ...

Radiotracer studies on protein metabolism in different focuses of growth in vivo

International Nuclear Information System (INIS)

Rapoport, E.A.; Konikova, A.S.

1979-01-01

The role of various components of protein turnover in the liver growth induced by partial hepatectomy or phenobarbital as well as in malignant tumours has been studied with a radiotracer method. The ratio of the utilized precursors of exo- and endogenous origin during the formation of protein in focuses of growth, is labile, depends on a number of factors and varies even in the subcellular structures of the cell nucleus. It is probable that due to their reutilization through transreactions the protein structural units have in some focuses of growth and during formation of some proteins a definite superiority over the free aminoacids. It is also shown that the release of proteins and their degradation products from Walker 256 carcinosarcoma and the reutilization of proteins and their degradation products by sarcoma M-1 contribute to the protein turnover in the tumours. 8author)

Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

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Haddy K S Fye

Full Text Available Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose

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

Science.gov (United States)

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

2016-01-01

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

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.

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 ...

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...

Molecular profiling in the treatment of colorectal cancer: focus on regorafenib

Directory of Open Access Journals (Sweden)

Yan Y

2015-10-01

Full Text Available Yiyi Yan, Axel Grothey Department of Medical Oncology, Mayo Clinic, Rochester, MN, USA Abstract: Metastatic colorectal cancer (mCRC is a highly heterogeneous disease. Its treatment outcome has been significantly improved over the last decade with the incorporation of biological targeted therapies, including anti-EGFR antibodies, cetuximab and panitumumab, and VEGF inhibitors, bevacizumab, ramucirumab, and aflibercept. The identification of predictive biomarkers has further improved the survival by accurately selecting patients who are most likely to benefit from these treatments, such as RAS mutation profiling for EGFR antibodies. Regorafenib is a multikinase inhibitor currently used as late line therapy for mCRC. The molecular and genetic markers associated with regorafenib treatment response are yet to be characterized. Here, we review currently available clinical evidence of mCRC molecular profiling, such as RAS, BRAF, and MMR testing, and its role in targeted therapies with special focus on regorafenib treatment. Keywords: metastatic colon cancer, targeted therapy, molecular profiling, regorafenib 

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

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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.

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)

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis

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Eroukova Veronika

2008-12-01

Full Text Available Abstract Background Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, ~4700 strains for increased sensitivity to paromomycin, which targets the process of protein synthesis. Results As expected, our analysis indicated that the majority of deletion strains (134 with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45, cellular component biogenesis and organization (28, DNA maintenance (21, transport (20, others (38 and unknown (39. These may represent minor cellular target sites (side-effects for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. Conclusion We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s.

Chemical-genetic profile analysis in yeast suggests that a previously uncharacterized open reading frame, YBR261C, affects protein synthesis.

Science.gov (United States)

Alamgir, Md; Eroukova, Veronika; Jessulat, Matthew; Xu, Jianhua; Golshani, Ashkan

2008-12-03

Functional genomics has received considerable attention in the post-genomic era, as it aims to identify function(s) for different genes. One way to study gene function is to investigate the alterations in the responses of deletion mutants to different stimuli. Here we investigate the genetic profile of yeast non-essential gene deletion array (yGDA, approximately 4700 strains) for increased sensitivity to paromomycin, which targets the process of protein synthesis. As expected, our analysis indicated that the majority of deletion strains (134) with increased sensitivity to paromomycin, are involved in protein biosynthesis. The remaining strains can be divided into smaller functional categories: metabolism (45), cellular component biogenesis and organization (28), DNA maintenance (21), transport (20), others (38) and unknown (39). These may represent minor cellular target sites (side-effects) for paromomycin. They may also represent novel links to protein synthesis. One of these strains carries a deletion for a previously uncharacterized ORF, YBR261C, that we term TAE1 for Translation Associated Element 1. Our focused follow-up experiments indicated that deletion of TAE1 alters the ribosomal profile of the mutant cells. Also, gene deletion strain for TAE1 has defects in both translation efficiency and fidelity. Miniaturized synthetic genetic array analysis further indicates that TAE1 genetically interacts with 16 ribosomal protein genes. Phenotypic suppression analysis using TAE1 overexpression also links TAE1 to protein synthesis. We show that a previously uncharacterized ORF, YBR261C, affects the process of protein synthesis and reaffirm that large-scale genetic profile analysis can be a useful tool to study novel gene function(s).

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.

Density profile measurements from a two-gun plasma focus device

International Nuclear Information System (INIS)

Tzeng, C.C.; Yen, C.K.; Yeh, T.R.; Kuo, Y.Y.; Shang, D.J.; Yu, Y.Z.; Hou, W.S.

1990-01-01

The dynamics of the plasma evolution in a two-gun plasma focus device has been studied using the laser shadowgraphy as well as the laser interferometry. The experiments were carried out from a 700 kJ two-gun plasma focus device reported earlier, which consisted of a pair of Mather type coaxial electrodes connected muzzle to muzzle. Previous results indicated that the simultaneous formation of the two deuterium plasma foci occurred earlier and then after ∼ 100 ns a disk-shaped plasma of ∼ 1.5 cm in diameter appeared in the middle region between the anodes. It is, therefore, the authors' goal to study the density profiles in the plasma foci and the middle region in order to understand further the formation of the plasma foci and their time evolution. The laser shadowgraphy was done with a XeCl excimer pumped dye laser system which operated at 550 nm with pulse width of ∼ 10 ns. The laser interferometry, on the other hand, was carried out using a TEA-TEA oscillator-amplifier N 2 -laser system with 337.1 nm and subnano-second pulse width. Both results show that the maximum electron density is ≥2 x 10 19 cm -3 and, in addition, the growth of the hydrodynamic instabilities are observed. These results together with the detailed density profiles are presented and discussed

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.

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

Energy Technology Data Exchange (ETDEWEB)

Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

2017-12-06

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

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.

Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

Czech Academy of Sciences Publication Activity Database

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

2015-01-01

Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

Accelerating Information Retrieval from Profile Hidden Markov Model Databases.

Directory of Open Access Journals (Sweden)

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.

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.

Signaling pathway-focused gene expression profiling in pressure overloaded hearts

Directory of Open Access Journals (Sweden)

Marco Musumeci

2011-01-01

Full Text Available The β-blocker propranolol displays antihypertrophic and antifibrotic properties in the heart subjected to pressure overload. Yet the underlying mechanisms responsible for these important effects remain to be completely understood. The purpose of this study was to determine signaling pathway-focused gene expression profile associated with the antihypertrophic action of propranolol in pressure overloaded hearts. To address this question, a focused real-time PCR array was used to screen left ventricular RNA expression of 84 gene transcripts representative of 18 different signaling pathways in C57BL/6 mice subjected to transverse aortic constriction (TAC or sham surgery. On the surgery day, mice received either propranolol (80 mg/kg/day or vehicle for 14 days. TAC caused a 49% increase in the left ventricular weight-to-body weight (LVW/BW ratio without changing gene expression. Propranolol blunted LVW/BW ratio increase by approximately 50% while causing about a 3-fold increase in the expression of two genes, namely Brca1 and Cdkn2a, belonging to the TGF-beta and estrogen pathways, respectively. In conclusion, after 2 weeks of pressure overload, TAC hearts show a gene expression profile superimposable to that of sham hearts. Conversely, propranolol treatment is associated with an increased expression of genes which negatively regulate cell cycle progression. It remains to be established whether a mechanistic link between gene expression changes and the antihypertrophic action of propranolol occurs.

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.

Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

NARCIS (Netherlands)

Bischoff, Rainer; Roecklin, D.; Roitsch, C.

1992-01-01

Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

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.

Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

Directory of Open Access Journals (Sweden)

Deborah R Boone

Full Text Available Cognitive deficits in survivors of traumatic brain injury (TBI are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive or surviving (Fluoro-Jade-negative pyramidal neurons obtained by laser capture microdissection (LCM. In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

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.

Science.gov (United States)

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

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.

Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation

NARCIS (Netherlands)

Padhan, N.; Yan, J.; Boge, A.; Scrivener, E.; Birgisson, H.; Zieba, A.; Gullberg, M.; Kamali-Moghaddam, M.; Claesson-Welsh, L.; Landegren, U.

2017-01-01

Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms

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/.

Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

Science.gov (United States)

Yang, Wen-Kai; Kang, Chao-Kai; Chang, Chia-Hao; Hsu, An-Di; Lee, Tsung-Han; Hwang, Pung-Pung

2013-01-01

FXYD proteins are novel regulators of Na+-K+-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for

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

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

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.

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 ...

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

Directory of Open Access Journals (Sweden)

Mosley R Lee

2008-09-01

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

Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

Science.gov (United States)

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

Protein profile of mouse ovarian follicles grown in vitro.

Science.gov (United States)

Anastácio, Amandine; Rodriguez-Wallberg, Kenny A; Chardonnet, Solenne; Pionneau, Cédric; Fédérici, Christian; Almeida Santos, Teresa; Poirot, Catherine

2017-12-01

Could the follicle proteome be mapped by identifying specific proteins that are common or differ between three developmental stages from the secondary follicle (SF) to the antrum-like stage? From a total of 1401 proteins identified in the follicles, 609 were common to the three developmental stages investigated and 444 were found uniquely at one of the stages. The importance of the follicle as a functional structure has been recognized; however, up-to-date the proteome of the whole follicle has not been described. A few studies using proteomics have previously reported on either isolated fully-grown oocytes before or after meiosis resumption or cumulus cells. The experimental design included a validated mice model for isolation and individual culture of SFs. The system was chosen as it allows continuous evaluation of follicle growth and selection of follicles for analysis at pre-determined developmental stages: SF, complete Slavjanski membrane rupture (SMR) and antrum-like cavity (AF). The experiments were repeated 13 times independently to acquire the material that was analyzed by proteomics. SFs (n = 2166) were isolated from B6CBA/F1 female mice (n = 42), 12 days old, from 15 l. About half of the follicles isolated as SF were analyzed as such (n = 1143) and pooled to obtain 139 μg of extracted protein. Both SMR (n = 359) and AF (n = 124) were obtained after individual culture of 1023 follicles in a microdrop system under oil, selected for analysis and pooled, to obtain 339 μg and 170 μg of protein, respectively. The follicle proteome was analyzed combining isoelectric focusing (IEF) fractionation with 1D and 2D LC-MS/MS analysis to enhance protein identification. The three protein lists were submitted to the 'Compare gene list' tool in the PANTHER website to gain insights on the Gene Ontology Biological processes present and to Ingenuity Pathway Analysis to highlight protein networks. A label-free quantification was performed with 1D LC-MS/MS analyses to

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.

Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

Science.gov (United States)

Xie, Song-Fang; Gao, Han; Niu, Li-Li; Xie, Zhen-Sheng; Fang, Fang; Wu, Zhi-Yong; Yang, Fu-Quan

2018-01-25

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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...

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.

Directory of Open Access Journals (Sweden)

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.

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.

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

Science.gov (United States)

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.

Focused ion-beam line profiles: A study of some factors affecting beam broadening

International Nuclear Information System (INIS)

Templeton, I.M.; Champion, H.G.

1995-01-01

The current--density profile of a focused ion beam (FIB) has a central peak accompanied by broader ''wings'' that, while unimportant in lithographic applications, can lead to unwanted effects during an implantation operation. The origin of the wings, and hence the best way to minimize them, is not clear and needs further study. We have measured the line profiles of several of the ions available in our FIB machine as a function of a number of variables, under ultrahigh vacuum (UHV) conditions. No effects are observed from changes in emission current or deliberate defocusing of the objective lens. There are some changes with beam aperture and/or current, but the biggest differences seem to be associated with a change of source type and hence, possibly, with a change in the source/extractor configuration or in the alloy and the emission process. The wing amplitudes are appreciably lower than many previously observed, and their profiles, at least for the lighter ions studied (Be ++ , Be + , and B + ), are Gaussian rather than exponential. It seems possible that our UHV conditions may have eliminated a scattering mechanism responsible for the larger, exponential wings previously observed. The corresponding beam and rectangle-edge profiles have been calculated. copyright 1995 American Vacuum Society

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

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

Science.gov (United States)

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.

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.

Real-time monitoring and chemical profiling of a cultivation process

DEFF Research Database (Denmark)

Mortensen, Peter P.; Bro, Rasmus

2006-01-01

they are known to reflect important properties of the fermentation process. Focus is also on important sampling issues-mainly structurally sub-optimal primary sampling methods affecting the representativity obtainable relative to the lot characteristics. Several different calibration approaches are investigated....... An enzyme marker profile as well as a tryptophan (protein marker) profile is identified. (c) 2006 Elsevier B.V All rights reserved....

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.

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

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 (

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.

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...

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.

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

Directory of Open Access Journals (Sweden)

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.

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.

Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply

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Laura eCeballos-Laita

2015-03-01

Full Text Available The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164 were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5% changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as

[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.

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...

Relativistic self-focusing of ultra-high intensity X-ray laser beams in warm quantum plasma with upward density profile

International Nuclear Information System (INIS)

Habibi, M.; Ghamari, F.

2014-01-01

The results of a numerical study of high-intensity X-ray laser beam interaction with warm quantum plasma (WQP) are presented. By means of an upward ramp density profile combined with quantum factors specially the Fermi velocity, we have demonstrated significant relativistic self-focusing (RSF) of a Gaussian electromagnetic beam in the WQP where the Fermi temperature term in the dielectric function is important. For this purpose, we have considered the quantum hydrodynamics model that modifies refractive index of inhomogeneous WQPs with the inclusion of quantum correction through the quantum statistical and diffraction effects in the relativistic regime. Also, to better illustration of the physical difference between warm and cold quantum plasmas and their effect on the RSF, we have derived the envelope equation governing the spot size of X-ray laser beam in Q-plasmas. In addition to the upward ramp density profile, we have found that the quantum effects would be caused much higher oscillation and better focusing of X-ray laser beam in the WQP compared to that of cold quantum case. Our computational results reveal the importance of the use of electrons density profile and Fermi speed in enhancing self-focusing of laser beam

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.

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...

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.

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

Directory of Open Access Journals (Sweden)

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.

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

Directory of Open Access Journals (Sweden)

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.

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...

Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

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Weber, Daniela; Davies, Michael J; Grune, Tilman

2015-08-01

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

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

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

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Goshima, Gohta; Nédélec, François; Vale, Ronald D

2005-10-24

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

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

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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.

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)

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.

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

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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.

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

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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.

Electrophoretic protein profiles of mid-sized copepod Calanoides patagoniensis steadily fed bloom-forming diatoms

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Victor M Aguilera

2015-09-01

Full Text Available Recent field and experimental evidence collected in the southern upwelling region off Concepción (36°5'S, 73°3'W showed an abrupt reduction (<72 h in the egg production rates (EPR of copepods when they were fed steadily and solely with the local bloom-forming diatom Thalassiosira rotula. Because diatoms were biochemically similar to dinoflagellate Prorocentrum minimum, a diet which supported higher reproductive outcomes, the fecundity reduction observed in copepod females fed with the diatom may have obeyed to post-ingestive processes, giving rise to resources reallocation. This hypothesis was tested by comparing feeding (clearance and ingestion rates, reproduction (EPR and hatching success and the structure of protein profiles (i.e., number and intensity of electrophoretic bands of copepods (adults and eggs incubated during 96 h with the two food conditions. The structure of protein profiles included molecular sizes that were calculated from the relative mobility of protein standards against the logarithm of their molecular sizes. After assessing the experimental conditions, feeding decreased over time for those females fed with T. rotula, while reproduction was higher in females fed with P. minimum. Electrophoretic profiles resulted similar mostly at a banding region of 100 to 89-kDa, while they showed partial differences around the region of 56-kDa band, especially in those females fed and eggs produced with T. rotula. Due to reproductive volume was impacted while larvae viability, a physiological processes with specific and high nutritional requirements, was independent on food type; post-ingestive processes, such as expression of stress-related proteins deviating resources to metabolic processes others than reproduction, are discussed under framework of nutritional-toxic mechanisms mediating copepod-diatoms relationships in productive upwelling areas.

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

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Camera, Donny M; Burniston, Jatin G; Pogson, Mark A; Smiles, William J; Hawley, John A

2017-12-01

It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise. © FASEB.

Exopolysaccharide-associated protein sorting in environmental organisms: the PEP-CTERM/EpsH system. Application of a novel phylogenetic profiling heuristic

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Ward Naomi

2006-08-01

Full Text Available Abstract Background Protein translocation to the proper cellular destination may be guided by various classes of sorting signals recognizable in the primary sequence. Detection in some genomes, but not others, may reveal sorting system components by comparison of the phylogenetic profile of the class of sorting signal to that of various protein families. Results We describe a short C-terminal homology domain, sporadically distributed in bacteria, with several key characteristics of protein sorting signals. The domain includes a near-invariant motif Pro-Glu-Pro (PEP. This possible recognition or processing site is followed by a predicted transmembrane helix and a cluster rich in basic amino acids. We designate this domain PEP-CTERM. It tends to occur multiple times in a genome if it occurs at all, with a median count of eight instances; Verrucomicrobium spinosum has sixty-five. PEP-CTERM-containing proteins generally contain an N-terminal signal peptide and exhibit high diversity and little homology to known proteins. All bacteria with PEP-CTERM have both an outer membrane and exopolysaccharide (EPS production genes. By a simple heuristic for screening phylogenetic profiles in the absence of pre-formed protein families, we discovered that a homolog of the membrane protein EpsH (exopolysaccharide locus protein H occurs in a species when PEP-CTERM domains are found. The EpsH family contains invariant residues consistent with a transpeptidase function. Most PEP-CTERM proteins are encoded by single-gene operons preceded by large intergenic regions. In the Proteobacteria, most of these upstream regions share a DNA sequence, a probable cis-regulatory site that contains a sigma-54 binding motif. The phylogenetic profile for this DNA sequence exactly matches that of three proteins: a sigma-54-interacting response regulator (PrsR, a transmembrane histidine kinase (PrsK, and a TPR protein (PrsT. Conclusion These findings are consistent with the hypothesis

Characterization of protein adsorption onto FePt nanoparticles using dual-focus fluorescence correlation spectroscopy

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Pauline Maffre

2011-07-01

Full Text Available Using dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces.

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

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Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix

2015-10-01

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

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Rotem Gura Sadovsky

2017-03-01

Full Text Available The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists.

Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

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Calvano, C D; Aresta, A; Iacovone, M; De Benedetto, G E; Zambonin, C G; Battaglia, M; Ditonno, P; Rutigliano, M; Bettocchi, C

2010-03-11

Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between

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

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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.

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

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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

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.

Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

DEFF Research Database (Denmark)

Albrethsen, J.; Kaas, A.; Schonle, E.

2009-01-01

Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably...... be examined for potential bias between sample groups. S ELDI-TOF MS protein profiling was used for preliminary evaluation of a biological-bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000......-2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected...

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

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SIWI PRAMATAMA MARS WIJAYANTI

2012-03-01

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

Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

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Sá-Pereira, Paula; Diniz, Mário S; Moita, Liliana; Pinheiro, Teresa; Mendonça, Elsa; Paixão, Susana M; Picado, Ana

2018-05-01

The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO 2 -NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO 2 -NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO 2 -NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO 2 -NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO 2 -NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO 2 -NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO 2 -NP toxicity in D. magna, providing useful information for future research.

Volatile profile, lipid oxidation and protein oxidation of irradiated ready-to-eat cured turkey meat products

International Nuclear Information System (INIS)

Feng, Xi; Ahn, Dong Uk

2016-01-01

Irradiation had little effects on the thiobarbituric acid reactive substances (TBARS) values in ready-to-eat (RTE) turkey meat products, while it increased protein oxidation at 4.5 kGy. The volatile profile analyses indicated that the amount of sulfur compounds increased linearly as doses increased in RTE turkey meat products. By correlation analysis, a positive correlation was found between benzene/ benzene derivatives and alcohols with lipid oxidation, while aldehydes, ketones and alkane, alkenes and alkynes were positively correlated with protein oxidation. Principle component analysis showed that irradiated meat samples can be discriminated by two categories of volatile compounds: Strecker degradation products and radiolytic degradation products. The cluster analysis of volatile data demonstrated that low-dose irradiation had minor effects on the volatile profile of turkey sausages (<1.5 kGy). However, as the doses increased, the differences between the irradiated and non-irradiated cured turkey products became significant. - Highlights: • Irradiation had little effects on lipid oxidation of ready-to-eat cured turkey. • 4.5 kGy irradiation increased protein oxidation. • Irradiated samples were isolated due to Strecker/radiolytic degradation products. • 1.5 kGy irradiation had limited effects on the volatile profile of turkey sausages. • Dimethyl disulfide can be used as a potential marker for irradiated meat products.

Detection of lung cancer using plasma protein profiling by matrix-assisted laser desorption/ionization mass spectrometry.

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Shevchenko, Valeriy E; Arnotskaya, Natalia E; Zaridze, David G

2010-01-01

There are no satisfactory plasma biomarkers which are available for the early detection and monitoring of lung cancer, one of the most frequent cancers worldwide. The aim of this study is to explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) to plasma proteomic patterns to distinguish lung cancer patients from healthy individuals. The EDTA plasma samples have been pre-fractionated using magnetic bead kits functionalized with weak cation exchange coatings. We compiled MS protein profiles for 90 patients with squamous cell carcinomas (SCC) and compared them with profiles from 187 healthy controls. The MALDI-ToF spectra were analyzed statistically using ClinProTools bioinformatics software. Depending on the sample used, up to 441 peaks/spectrum could be detected in a mass range of 1000-20,000 Da; 33 of these proteins had statistically differential expression levels between SCC and control plasma (P 90%) in external validation test. These results suggest that plasma MALDI-ToF MS protein profiling can distinguish patients with SCC and also from healthy individuals with relatively high sensitivity and specificity and that MALDI- ToF MS is a potential tool for the screening of lung cancer.

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

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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.

Green Fluorescent Protein-Focused Bioinformatics Laboratory Experiment Suitable for Undergraduates in Biochemistry Courses

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Rowe, Laura

2017-01-01

An introductory bioinformatics laboratory experiment focused on protein analysis has been developed that is suitable for undergraduate students in introductory biochemistry courses. The laboratory experiment is designed to be potentially used as a "stand-alone" activity in which students are introduced to basic bioinformatics tools and…

ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS

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Kavita Rajesh Pandey

2016-09-01

Full Text Available Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII obtained in previous studies (Dubey and Vakil, 2010, were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell.

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

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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

Strain-dependent profile of misfolded prion protein aggregates.

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

2016-02-15

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

Predicting adverse drug reaction profiles by integrating protein interaction networks with drug structures.

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Huang, Liang-Chin; Wu, Xiaogang; Chen, Jake Y

2013-01-01

The prediction of adverse drug reactions (ADRs) has become increasingly important, due to the rising concern on serious ADRs that can cause drugs to fail to reach or stay in the market. We proposed a framework for predicting ADR profiles by integrating protein-protein interaction (PPI) networks with drug structures. We compared ADR prediction performances over 18 ADR categories through four feature groups-only drug targets, drug targets with PPI networks, drug structures, and drug targets with PPI networks plus drug structures. The results showed that the integration of PPI networks and drug structures can significantly improve the ADR prediction performance. The median AUC values for the four groups were 0.59, 0.61, 0.65, and 0.70. We used the protein features in the best two models, "Cardiac disorders" (median-AUC: 0.82) and "Psychiatric disorders" (median-AUC: 0.76), to build ADR-specific PPI networks with literature supports. For validation, we examined 30 drugs withdrawn from the U.S. market to see if our approach can predict their ADR profiles and explain why they were withdrawn. Except for three drugs having ADRs in the categories we did not predict, 25 out of 27 withdrawn drugs (92.6%) having severe ADRs were successfully predicted by our approach. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma Protein Profiles Differ Between Women Diagnosed with Cervical Intraepithelial Neoplasia (CIN 1 and 3

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Edward E. Partridge

2006-01-01

Full Text Available Early detection of precancerous cells in the cervix and their clinical management is the main purpose of cervical cancer prevention and treatment programs. Cytological findings or testing for high risk (HR-human papillomavirus (HPV are inadequately sensitive for use in triage of women at high risk for cervical cancer. The current study is an exploratory study to identify candidate surface-enhanced laser desorption/ionization (SELDI time of flight (TOF mass spectrometry (MS protein profiles in plasma that may distinguish cervical intraepithelial neoplasia (CIN 3 from CIN 1 among women infected with HR-HPV. We evaluated the SELDI-TOF-MS plasma protein profiles of HR-HPV positive 32 women with CIN 3 (cases and 28 women with CIN1 (controls. Case-control status was kept blinded and triplicates of each sample and quality control plasma samples were randomized and after robotic sample preparations were run on WCX2 chips. After alignment of mass/charge (m-z values, an iterative method was used to develop a classifier on a training data set that had 28 cases and 22 controls. The classifier developed was used to classify the subjects in a test data set that has six cases and six controls. The classifier separated the cases from controls in the test set with 100% sensitivity and 100% specificity suggesting the possibility of using plasma SELDI protein profiles to identify women who are likely to have CIN 3 lesions.

Protein Profile in Corpus Luteum during Pregnancy in Korean Native Cows

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H. J. Chung

2012-11-01

Full Text Available Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5 and pregnant cows (n = 6; until d-90. A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each. Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4 was compared with that in CL of cyclic cows at d 6 to 13 (n = 5. Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE, and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS. Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows.

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling

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Johana A. Luna Coronell

2018-02-01

Full Text Available Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 “CRC genes.” These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology. Keywords: Autoantibody tumor biomarker, Cancer immunology, Colorectal cancer, Immunomics, Protein microarray

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

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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.

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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.

Dietary Protein Intake in Dutch Elderly People: A Focus on Protein Sources

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Michael Tieland

2015-11-01

Full Text Available Introduction: Sufficient high quality dietary protein intake is required to prevent or treat sarcopenia in elderly people. Therefore, the intake of specific protein sources as well as their timing of intake are important to improve dietary protein intake in elderly people. Objectives: to assess the consumption of protein sources as well as the distribution of protein sources over the day in community-dwelling, frail and institutionalized elderly people. Methods: Habitual dietary intake was evaluated using 2- and 3-day food records collected from various studies involving 739 community-dwelling, 321 frail and 219 institutionalized elderly people. Results: Daily protein intake averaged 71 ± 18 g/day in community-dwelling, 71 ± 20 g/day in frail and 58 ± 16 g/day in institutionalized elderly people and accounted for 16% ± 3%, 16% ± 3% and 17% ± 3% of their energy intake, respectively. Dietary protein intake ranged from 10 to 12 g at breakfast, 15 to 23 g at lunch and 24 to 31 g at dinner contributing together over 80% of daily protein intake. The majority of dietary protein consumed originated from animal sources (≥60% with meat and dairy as dominant sources. Thus, 40% of the protein intake in community-dwelling, 37% in frail and 29% in institutionalized elderly originated from plant based protein sources with bread as the principle source. Plant based proteins contributed for >50% of protein intake at breakfast and between 34% and 37% at lunch, with bread as the main source. During dinner, >70% of the protein intake originated from animal protein, with meat as the dominant source. Conclusion: Daily protein intake in these older populations is mainly (>80% provided by the three main meals, with most protein consumed during dinner. More than 60% of daily protein intake consumed is of animal origin, with plant based protein sources representing nearly 40% of total protein consumed. During dinner, >70% of the protein intake originated from

A comparative analysis of phloem exudate proteins from Cucumis melo, Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing.

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Sabnis, D D; Hart, J W

1976-01-01

Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.

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.

Effect of Heating Method on Alteration of Protein Molecular Structure in Flaxseed: Relationship with Changes in Protein Subfraction Profile and Digestion in Dairy Cows.

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Ahmad Khan, Nazir; Booker, Helen; Yu, Peiqiang

2015-02-04

This study evaluated the effect of heating methods on alteration of protein molecular structure in flaxseed (Linum usitatissimum L.) in relation to changes in protein subfraction profile and digestion in dairy cows. Seeds from two flaxseed varieties, sampled from two replicate plots at two locations, were evaluated. The seeds were either maintained in their raw state or heated in an air-draft oven (dry heating) or autoclave (moist heating) for 60 min at 120 °C or by microwave irradiation (MIR) for 5 min. Compared to raw seeds, moist heating decreased (P RUP) content (36.0 ± 5.19 to 46.9 ± 2.72% CP) and intestinal digestibility of RUP (61.0 ± 2.28 to 63.8 ± 2.67% RUP). Dry heating did not alter (P > 0.05) the protein subfraction profile and rumen degradation kinetics, whereas MIR increased (P RUP content from 36.0 ± 5.19 to 40.4 ± 4.67% CP. The MIR and dry heating did not alter (P > 0.05) the amide I to amide II ratio, but moist heating decreased (P RUP (R 2 = 0.71), and intestinal digestibility of RUP (R 2 = 0.72). Overall, heat-induced changes in protein nutritive value and digestion were strongly associated with heat-induced alteration in protein molecular structures.

Combining RNA-seq and proteomic profiling to identify seminal fluid proteins in the migratory grasshopper Melanoplus sanguinipes (F).

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Bonilla, Martha L; Todd, Christopher; Erlandson, Martin; Andres, Jose

2015-12-22

Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America. Using RNA sequencing, we characterized the transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs' functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species- specific genetic control strategies.

Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction

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Wang, Jing; Ma, Zihao; Carr, Steven A.; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W.; Ellis, Matthew J. C.; Townsend, R. Reid; Smith, Richard D.; McDermott, Jason E.; Chen, Xian; Paulovich, Amanda G.; Boja, Emily S.; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Rodland, Karin D.; Liebler, Daniel C.; Zhang, Bing

2016-11-11

Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies

Consequences of occupational food-related hand dermatoses with a focus on protein contact dermatitis

DEFF Research Database (Denmark)

Vester, Lotte; Thyssen, Jacob P; Menné, Torkil

2012-01-01

Background. Protein contact dermatitis is a frequent disorder among hand eczema patients who have occupational food contact. Knowledge about the consequences of having protein contact dermatitis is lacking. Objectives. To investigate the consequences of having occupational skin disease on the hands...... resulting from food handling, with a focus on protein contact dermatitis. Material and methods. One hundred and seventy-eight patients who were identified as having skin disease related to occupational food exposure and who answered a questionnaire concerning the consequences of their skin disease were......%, respectively, of the patients with other occupational food-related hand dermatoses (p = 0.02). Sixty-two per cent and 43%, respectively, had to change job because of skin problems (p = 0.02). Atopic dermatitis was equally common in the two groups. Conclusion. We found that the patients with protein contact...

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.

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...

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/.

Fabrication of nested elliptical KB mirrors using profile coating for synchrotron radiation X-ray focusing

International Nuclear Information System (INIS)

Liu Chian; Ice, G.E.; Liu, W.; Assoufid, L.; Qian, J.; Shi, B.; Khachatryan, R.; Wieczorek, M.; Zschack, P.; Tischler, J.Z.

2012-01-01

This paper describes fabrication methods used to demonstrate the advantages of nested or Montel optics for micro/nanofocusing of synchrotron X-ray beams. A standard Kirkpatrick-Baez (KB) mirror system uses two separated elliptical mirrors at glancing angles to the X-ray beam and sequentially arranged at 90° to each other to focus X-rays successively in the vertical and horizontal directions. A nested KB mirror system has the two mirrors positioned perpendicular and side-by-side to each other. Compared to a standard KB mirror system, Montel optics can focus a larger divergence and the mirrors can have a shorter focal length. As a result, nested mirrors can be fabricated with improved demagnification factor and ultimately smaller focal spot, than with a standard KB arrangement. The nested system is also more compact with an increased working distance, and is more stable, with reduced complexity of mirror stages. However, although Montel optics is commercially available for laboratory X-ray sources, due to technical difficulties they have not been used to microfocus synchrotron radiation X-rays, where ultra-precise mirror surfaces are essential. The main challenge in adapting nested optics for synchrotron microfocusing is to fabricate mirrors with a precise elliptical surface profile at the very edge where the two mirrors meet and where X-rays scatter. For example, in our application to achieve a sub-micron focus with high efficiency, a surface figure root-mean-square (rms) error on the order of 1 nm is required in the useable area along the X-ray footprint with a ∼0.1 mm-diameter cross section. In this paper we describe promising ways to fabricate precise nested KB mirrors using our profile coating technique and inexpensive flat Si substrates.

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

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Muhammad Ibrahim

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

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

Science.gov (United States)

Ibrahim, Muhammad; Shi, Yu; Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Blondel, Carlos; Santiviago, Carlos A; Contreras, Ines; Sun, Guochang

2012-01-01

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

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

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Schlarman Maggie S

2012-03-01

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

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.

Optimizing UV laser focus profiles for improved MALDI performance.

Science.gov (United States)

Holle, Armin; Haase, Andreas; Kayser, Markus; Höhndorf, Jens

2006-06-01

Matrix assisted laser desorption/ionization (MALDI) applications, such as proteomics, genomics, clinical profiling and MALDI imaging, have created a growing demand for faster instrumentation. Since the commonly used nitrogen lasers have throughput and life span limitations, diode-pumped solid-state lasers are an alternative. Unfortunately this type of laser shows clear performance limitations in MALDI in terms of sensitivity, resolution and ease of use, for applications such as thin-layer sample preparations, acceptance of various matrices (e.g. DHB for glycopeptides) and MALDI imaging. While it is obvious that the MALDI process has some dependence on the characteristics of the laser used, it is unclear which features are the most critical in determining laser performance for MALDI. In this paper we show, for the first time, that a spatially structured laser beam profile in lieu of a Gaussian profile is of striking importance. This result enabled us to design diode-pumped Nd : YAG lasers that on various critical applications perform as well for MALDI as the nitrogen lasers and in some respects even better. The modulation of the beam profile appears to be a new parameter for optimizing the MALDI process. In addition, the results trigger new questions directing us to a better understanding of the MALDI process. Copyright (c) 2006 John Wiley & Sons, Ltd.

Integrated transcriptomics and proteomics analysis of storage protein composition in developing barley grain to improve nutritional profile

DEFF Research Database (Denmark)

Kaczmarczyk, Agnieszka Ewa; Dionisio, Giuseppe; Renaut, Jenny

2012-01-01

The aim of the study was to understand the molecular and biochemical mechanisms underpinning the effect of nitrogen (N) on barley (Hordeum vulgare) storage protein production (hordeins) during grain filling. Using a combination of advanced biochemistry methods, we could comprehensively describe......-regimes caused significant differences in both quantity and quality of the storage proteins transcripts. Principal Component Analysis of the amino acid (AA) profiles also indicated dissimilarity in individual AA percentages, correlated to hordein content. The abundance values of proteins of interest confirmed...

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

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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

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....

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

Science.gov (United States)

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.

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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Yuping Ren

2017-12-01

Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

Examination on the protein profiles of salivary glands of P. berghei infected anopheles Sp. post gamma irradiation using SDS-PAGE technique for developing malaria vaccine

International Nuclear Information System (INIS)

Tetriana, D.; Syaifudin, M.

2014-01-01

Sporozoite is a step of malaria parasitic live cycle that is most invasive and appropriate vaccine candidate. Result of experiments showed that malaria vaccine created by attenuating Plasmodium sp sporozoites with gamma rays was proven more effective. Study on the effects of irradiation to the profiles of protein in vaccine development is also important. The aim of this research was to examine the protein profile of salivary glands in sporozoite infected Anopheles sp post gamma irradiation using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. Examination covered the infection of Anopheles sp with Plasmodium sp, maintenance of infected mosquitoes for 14-16 days to obtain sporozoites, in vivo - in vitro irradiation of mosquitoes, preparation of salivary glands, electrophoresis on 10% SDS-PAGE, and Commassie blue staining. Results showed a different protein profile of infected and non infected salivary glands of Anopheles sp. There was additional protein band numbers at higher dose of irradiation (200 Gy) from sporozoite protein of P. berghei (MW 62 kDa). However, no difference of the profiles of circumsporozoite protein (CSP) observed among gamma irradiation doses of 150, 175 and 200 Gy. These results provide basic information that would lead to further study on the role of sporozoite proteins in malaria vaccine development. (author)

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

DEFF Research Database (Denmark)

Stevenson, L.S.; McCullough, K.; Vincent, I.

2006-01-01

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

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

Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

Science.gov (United States)

2014-03-10

leishmaniasis.56 Pre-exposure of PROFILING OF SAND FLY SALIVARY PROTEINS 935 murine cells to L. intermedia salivary sonicates resulted in decreased IP-10...Thompson JD, Higgins DG, 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7...Brodskyn C, Barral A, de Oliveira CI, 2010. Immunity to Lutzomyia intermedia saliva modulates the inflammatory environ- ment induced by Leishmania

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

DEFF Research Database (Denmark)

Beuchert Kallehauge, Thomas; Li, Shangzhong; Pedersen, Lasse Ebdrup

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as effici......Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated...... as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated...... as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we...

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

Science.gov (United States)

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

2013-11-01

Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

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.

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.

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.

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.

Proteoform profiling of peripheral blood serum proteins from pregnant women provides a molecular IUGR signature.

Science.gov (United States)

Wölter, M; Röwer, C; Koy, C; Rath, W; Pecks, U; Glocker, M O

2016-10-21

Intrauterine growth restriction (IUGR) is an important cause of perinatal morbidity and mortality and contributes substantially to medically indicated preterm birth; preventing fetal death. Molecular profiling of the mothers' peripheral blood was desired to monitor the health conditions of the fetuses. To develop such a minimally invasive assay, we applied a protein affinity fractionation method to peripheral blood serum samples from pregnant women belonging to either the IUGR or to the control group. Proof-of-principle was shown by relative quantitation analysis of mixtures of intact proteoforms using MALDI-ToF mass spectrometry. The two best differentiating proteins and proteoforms, respectively, were apolipoprotein C-II and apolipoprotein C-III 0 . Together with three robustly expressed protein proteoforms proapolipoprotein C-II, apolipoprotein C-III 1 , and apolipoprotein C-III 2 , which served as landmarks for relative quantitation analysis, they constituted the maternal IUGR proteome signature. Separation confidence of our IUGR proteoform signature reached a sensitivity of 0.73 and a specificity of 0.87 with an area under curve of 0.86 in receiver operator characteristics. Identification of IUGR newborns in the case room is required as children are severely diseased and need specialized care during infancy. Yet, at time of birth there is no readily applicable clinical test available. Hence, a molecular profiling assay is highly desired. It needs to be mentioned that current clinical definitions and recommendations for IUGR are unfortunately misleading and are not universally applicable. The most commonly adopted definition is an abdominal circumference (AC) or estimated fetal weight measurement protein composition (IUGR signature) which can be determined just ahead of delivery and at date of delivery, respectively using a minimal invasive blood sampling approach. With this manuscript we describe the use of a mass spectrometric profiling method of 30

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

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Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

2015-01-01

Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (Pprostatitis patients from HV (Pprostatitis patients compared to HV (Pprostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

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

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

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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

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...

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.

Distinct expression profiles and different functions of odorant binding proteins in Nilaparvata lugens Stål.

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Peng He

Full Text Available BACKGROUND: Odorant binding proteins (OBPs play important roles in insect olfaction. The brown planthopper (BPH, Nilaparvata lugens Stål (Delphacidae, Auchenorrhyncha, Hemiptera is one of the most important rice pests. Its monophagy (only feeding on rice, wing form (long and short wing variation, and annual long distance migration (seeking for rice plants of high nutrition imply that the olfaction would play a central role in BPH behavior. However, the olfaction related proteins have not been characterized in this insect. METHODOLOGY/PRINCIPAL FINDINGS: Full length cDNA of three OBPs were obtained and distinct expression profiles were revealed regarding to tissue, developmental stage, wing form and gender for the first time for the species. The results provide important clues in functional differentiation of these genes. Binding assays with 41 compounds demonstrated that NlugOBP3 had markedly higher binding ability and wider binding spectrum than the other two OBPs. Terpenes and Ketones displayed higher binding while Alkanes showed no binding to the three OBPs. Focused on NlugOBP3, RNA interference experiments showed that NlugOBP3 not only involved in nymph olfaction on rice seedlings, but also had non-olfactory functions, as it was closely related to nymph survival. CONCLUSIONS: NlugOBP3 plays important roles in both olfaction and survival of BPH. It may serve as a potential target for developing behavioral disruptant and/or lethal agent in N. lugens.

Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins.

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Khan, Sheraz; Iqbal, Mazhar; Tariq, Muhammad; Baig, Shahid M; Abbas, Wasim

2018-01-01

HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

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Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Changes in the IgE-reacting protein profiles of Acer negundo, Platanus x acerifolia and Quercus robur pollen in response to ozone treatment.

Science.gov (United States)

Ribeiro, Helena; Duque, Laura; Sousa, Raquel; Cruz, Ana; Gomes, Carlos; da Silva, Joaquim Esteves; Abreu, Ilda

2014-01-01

This study aims to investigate the effects of O3 in protein content and immunoglobulin E (IgE)-binding profiles of Acer negundo, Platanus x acerifolia and Quercus robur pollen. Pollen was exposed to O3 in an environmental chamber, at half, equal and four times the limit value for the human health protection in Europe. Pollen total soluble protein was determined with Coomassie Protein Assay Reagent, and the antigenic and allergenic properties were investigated by SDS-PAGE and immunological techniques using patients' sera. O3 exposure affected total soluble protein content and some protein species within the SDS-PAGE protein profiles. Most of the sera revealed increased IgE reactivity to proteins of A. negundo and Q. robur pollen exposed to the pollutant compared with the non-exposed one, while the opposite was observed in P. x acerifolia pollen. So, the modifications seem to be species dependent, but do not necessarily imply that increase allergenicity would occur in atopic individuals.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

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de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

2017-08-10

Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open

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

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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

Protein profiling of preeclampsia placental tissues.

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

2014-01-01

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

Dietary protein intake in Dutch elderly people : a focus on protein sources

NARCIS (Netherlands)

Tieland, Michael; Borgonjen-Van den Berg, Karin J.; Van Loon, Luc J. C.; de Groot, Lisette C. P. G. M.

2015-01-01

INTRODUCTION: Sufficient high quality dietary protein intake is required to prevent or treat sarcopenia in elderly people. Therefore, the intake of specific protein sources as well as their timing of intake are important to improve dietary protein intake in elderly people. OBJECTIVES: to assess the

Dietary protein intake in Dutch elderly people: a focus on protein sources

NARCIS (Netherlands)

Tieland, C.A.B.; Borgonjen-van den Berg, K.J.; Loon, van L.J.C.; Groot, de C.P.G.M.

2015-01-01

Introduction: Sufficient high quality dietary protein intake is required to prevent or treat sarcopenia in elderly people. Therefore, the intake of specific protein sources as well as their timing of intake are important to improve dietary protein intake in elderly people. Objectives: to assess the

Deciphering Phosphotyrosine-Dependent Signaling Networks in Cancer by SH2 Profiling

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Machida, Kazuya; Khenkhar, Malik

2012-01-01

It has been a decade since the introduction of SH2 profiling, a modular domain-based molecular diagnostics tool. This review covers the original concept of SH2 profiling, different analytical platforms, and their applications, from the detailed analysis of single proteins to broad screening in translational research. Illustrated by practical examples, we discuss the uniqueness and advantages of the approach as well as its limitations and challenges. We provide guidance for basic researchers and oncologists who may consider SH2 profiling in their respective cancer research, especially for those focusing on tyrosine phosphoproteomics. SH2 profiling can serve as an alternative phosphoproteomics tool to dissect aberrant tyrosine kinase pathways responsible for individual malignancies, with the goal of facilitating personalized diagnostics for the treatment of cancer. PMID:23226573

The role of drug profiles as similarity metrics: applications to repurposing, adverse effects detection and drug-drug interactions.

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Vilar, Santiago; Hripcsak, George

2017-07-01

Explosion of the availability of big data sources along with the development in computational methods provides a useful framework to study drugs' actions, such as interactions with pharmacological targets and off-targets. Databases related to protein interactions, adverse effects and genomic profiles are available to be used for the construction of computational models. In this article, we focus on the description of biological profiles for drugs that can be used as a system to compare similarity and create methods to predict and analyze drugs' actions. We highlight profiles constructed with different biological data, such as target-protein interactions, gene expression measurements, adverse effects and disease profiles. We focus on the discovery of new targets or pathways for drugs already in the pharmaceutical market, also called drug repurposing, in the interaction with off-targets responsible for adverse reactions and in drug-drug interaction analysis. The current and future applications, strengths and challenges facing all these methods are also discussed. Biological profiles or signatures are an important source of data generation to deeply analyze biological actions with important implications in drug-related studies. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Protein Profiling of Preeclampsia Placental Tissues

Science.gov (United States)

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

2014-01-01

Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia. PMID:25392996

Protein profiling of preeclampsia placental tissues.

Directory of Open Access Journals (Sweden)

Chang Shu

Full Text Available Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A and adverse outcomes (Flt-1 in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

Response to DNA damage: why do we need to focus on protein phosphatases?

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Midori eShimada

2013-01-01

Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

Energy Technology Data Exchange (ETDEWEB)

Coquelle, Nicolas [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Brewster, Aaron S. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Kapp, Ulrike; Shilova, Anastasya; Weinhausen, Britta [European Synchrotron Radiation Facility, BP 220, 38043 Grenoble (France); Burghammer, Manfred, E-mail: burgham@esrf.fr [European Synchrotron Radiation Facility, BP 220, 38043 Grenoble (France); Ghent University, Ghent B-9000 (Belgium); Colletier, Jacques-Philippe, E-mail: burgham@esrf.fr [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France)

2015-05-01

A raster scanning serial protein crystallography approach is presented, that consumes as low ∼200–700 nl of sedimented crystals. New serial data pre-analysis software, NanoPeakCell, is introduced. High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

Profile measurement of a bent neutron mirror using an ultrahigh precision non-contact measurement system with an auto focus laser probe

International Nuclear Information System (INIS)

Morita, S; Guo, J; Yamagata, Y; Yamada, N L; Torikai, N; Takeda, S; Furusaka, M

2016-01-01

A bent neutron mirror has been considered as one of the best solutions for focusing neutron beams from the viewpoint of cost-benefit performance. Although the form deviation of the bent profile is expected because of the large spot size, it is difficult to measure due to its geometric limitation. Here, we propose a non-contact measurement system using an auto focus (AF) laser probe on an ultrahigh precision machine tool to precisely evaluate the form deviation of the bent mirror. The AF laser probe is composed of a diode laser, a position sensitive sensor, a charge-coupled device (CCD) camera and a microscope objective lens which is actuated by an electromagnetic motor with 1 nm resolution for position sensing and control. The sensor enables a non-contact profile measurement of a high precision surface without any surface damage in contrast with contact-type ultrahigh precision coordinate measurement machines with ruby styli. In the on-machine measurement system, a personal computer simultaneously acquires a displacement signal from the AF laser probe and 3-axis positional coordinates of the ultrahigh machine tool branched between the linear laser scales and the numerical controller. The acquisition rate of the 4-axis positional data in 1 nm resolution is more than 10 Hz and the simultaneity between the axes is negligible. The profile of a neutron bent mirror was measured from a transparent side using the developed system, and the result proves that the form deviation of the mirror enlarged the the spot size of focused neuron beam. (paper)

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

Science.gov (United States)

Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

2015-09-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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.

The importance of Pharmacovigilance for the drug safety: Focus on cardiovascular profile of incretin-based therapy.

Science.gov (United States)

Sportiello, Liberata; Rafaniello, Concetta; Scavone, Cristina; Vitale, Cristiana; Rossi, Francesco; Capuano, Annalisa

2016-01-01

With the recent introduction of the new European Pharmacovigilance legislation, all new drugs must be carefully monitored after admission on the European market, in order to assess the long safety profile. Currently, special attention is given to several hypoglycemic agents with recent market approval (agonists of glucagon-like peptide-1 [GLP-1] receptor and dipeptidyl peptidase 4 inhibitors [DPP-4i]), which act through the potentiation of incretin hormone signaling. Their inclusion in European additional monitoring is also due to safety problems, which seem to characterize their pharmacological class. In fact, these drugs initially showed a good tolerability profile with mainly gastrointestinal adverse events, low risk of hypoglycemia and minor effects on body weight. But, new concerns such as infections, pancreatitis, pancreatic cancer and above all cardiovascular events (especially risk of heart failure requiring hospitalization) are now arising. In this review, we highlighted aspects of the new Pharmacovigilance European dispositions, and then we investigated the tolerability profile of incretin-based therapies, in particular DPP-4 inhibitors. Notably, we focused our attention on new safety concerns, which are emerging mostly in the post-marketing period, as the cardiovascular risk profile. Evidence in literature and opinions of regulatory agencies (e.g., European Medicines Agency and Food and Drug Administration) about risks of incretin-based therapies are yet controversial, and there are many open questions in particular on cancer and cardiovascular effects. Thus, it is important to continue to monitor closely the use of these drugs in clinical practice to improve the knowledge on their long-term safety and their place in diabetes therapy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Divergent flow isoelectric focusing: fast and efficient method for protein sample preparation for mass spectrometry

Czech Academy of Sciences Publication Activity Database

Mazanec, Karel; Bobálová, Janette; Šlais, Karel

2009-01-01

Roč. 393, 6-7 (2009), s. 1769-1778 ISSN 1618-2642 R&D Projects: GA MŠk 1M0570; GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : Isoelectric focusing * proteins * MALDI-TOF/TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.480, year: 2009

Absorption of Vitamin A and Carotenoids by the Enterocyte: Focus on Transport Proteins

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Emmanuelle Reboul

2013-09-01

Full Text Available Vitamin A deficiency is a public health problem in most developing countries, especially in children and pregnant women. It is thus a priority in health policy to improve preformed vitamin A and/or provitamin A carotenoid status in these individuals. A more accurate understanding of the molecular mechanisms of intestinal vitamin A absorption is a key step in this direction. It was long thought that β-carotene (the main provitamin A carotenoid in human diet, and thus all carotenoids, were absorbed by a passive diffusion process, and that preformed vitamin A (retinol absorption occurred via an unidentified energy-dependent transporter. The discovery of proteins able to facilitate carotenoid uptake and secretion by the enterocyte during the past decade has challenged established assumptions, and the elucidation of the mechanisms of retinol intestinal absorption is in progress. After an overview of vitamin A and carotenoid fate during gastro-duodenal digestion, our focus will be directed to the putative or identified proteins participating in the intestinal membrane and cellular transport of vitamin A and carotenoids across the enterocyte (i.e., Scavenger Receptors or Cellular Retinol Binding Proteins, among others. Further progress in the identification of the proteins involved in intestinal transport of vitamin A and carotenoids across the enterocyte is of major importance for optimizing their bioavailability.

PanCoreGen - Profiling, detecting, annotating protein-coding genes in microbial genomes.

Science.gov (United States)

Paul, Sandip; Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V; Chattopadhyay, Sujay

2015-12-01

A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing the pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen - a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for a species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars - Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. Copyright © 2015 Elsevier Inc. All rights reserved.

A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics.

Science.gov (United States)

Burke, Kelly S; Parul, Dzmitry; Reddish, Michael J; Dyer, R Brian

2013-08-07

We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.

Effects of the dietary amount and source of protein, resistance training and anabolic-androgenic steroids on body weight and lipid profile of rats.

Science.gov (United States)

Aparicio, V A; Sánchez, C; Ortega, F B; Nebot, E; Kapravelou, G; Porres, J M; Aranda, P

2013-01-01

Dietary protein amount and source, hypertrophy resistance training (RT) and anabolicandrogenic steroids (AAS) may affect body weight and plasma and hepatic lipid profile. 157 adult male Wistar rats were randomly distributed in 16 experimental groups resulting in: normal-protein (NP) or high-protein (HP) diets, whey or soy-protein diets, with or without RT and with or without AAS, for 3 months. Final body weight was lower in the RT and AAS groups compared to sedentary and non- AAS groups, respectively (all, pweight of rats that performed RT or ingested a HP diet (all, p<0.05). HDL-cholesterol was higher when RT was combined with HP diets (p=0.010) or non-AAS and when HP diets were combined with non-AAS (both,p<0.001). Groups that combined RT with non-AAS administration obtained the lowest hepatic TAG (p<0.05). Among all the interventions tested, AAS was the factor that most negatively affected plasma and hepatic lipid profile, whereas HP diets and RT could benefit lipid profile, especially when combined. Copyright © AULA MEDICA EDICIONES 2013. Published by AULA MEDICA. All rights reserved.

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.

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.

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

Science.gov (United States)

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.

Molecular spectroscopic features of protein in newly developed chickpea: Relationship with protein chemical profile and metabolism in the rumen and intestine of dairy cows.

Science.gov (United States)

Sun, Baoli; Khan, Nazir Ahmad; Yu, Peiqiang

2018-05-05

The first aim of this study was to investigate the nutritional value of crude protein (CP) in CDC [Crop Development Centre (CDC), University of Saskatchewan] chickpea varieties (Frontier kabuli and Corinne desi) in comparison with a CDC barley variety in terms of: 1) CP chemical profile and subfractions; (2) in situ rumen degradation kinetics and intestinal digestibility of CP; 2) metabolizable protein (MP) supply to dairy cows; and (3) protein molecular structure characteristics using advanced molecular spectroscopy. The second aim was to quantify the relationship between protein molecular spectral characteristics and CP subfractions, in situ rumen CP degradation characteristics, intestinal digestibility of CP, and MP supply to dairy cows. Samples (n=4) of each variety, from two consecutive years were analyzed. Chickpeas had higher (Pmolecular spectral data of chickpeas can be distinguished from the barley. The two chickpeas did not differ in CP content, and any of the measured in situ degradation and molecular spectral characteristics of protein. The content of RUP was positively (r=0.94, Pmolecular spectroscopy can be used to rapidly characterize feed protein molecular structures and predict their digestibility and nutritive value. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

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

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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.

Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

Science.gov (United States)

Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

2016-04-01

Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; 36 weeks) preeclampsia and their controls who delivered preterm (n = 5; 36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

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...

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

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Laura Camacho

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

Protein Profiling of Preeclampsia Placental Tissues

OpenAIRE

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

2014-01-01

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

Study of protein and metabolic profile of sugarcane workers

Energy Technology Data Exchange (ETDEWEB)

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)

Economic impact profiling of CBRN events: focusing on biological incidents.

Science.gov (United States)

Cavallini, Simona; Bisogni, Fabio; Mastroianni, Marco

2014-12-01

Chemical, biological, radiological and nuclear (CBRN) incidents, both caused accidentally by human error or natural/technological events and determined intentionally as criminal/malicious/terroristic acts, have consequences that could be differently characterized. In the last years many efforts to analyze the economic impact of terrorist threat have been carried out, while researches specifically concerning CBRN events have not been extensively undertaken. This paper in particular aims at proposing a methodological approach for studying macro-level economic impact profiles of biological incidents caused by weaponized and non-weaponized materials. The suggested approach investigates the economic consequences of biological incidents according to two main dimensions: type of large-scale effect and persistence of effect. Biological incident economic impacts are analyzed taking into account the persistence of effect during time as short-term impact (i.e. immediately after the incident), medium-term impact (i.e. by a month) and long-term impact (i.e. by years). The costs due to preventive countermeasure against biological threats (e.g. prevention, protection and preparedness expenses) are not taken into account. To this purpose, information on the key features of past biological incidents can be used as case studies to try to build impact profiles taking into account the proposed two main dimensions. Consequence management and effect mitigation of CBRN emergencies and disasters may benefit from an ex ante definition of the impact profiling related to this kind of incidents. The final goal of this paper is to define an approach to organize information on possible biological events according to their impact profile for supporting more effective and efficient first responders' prompt actions and policy makers' strategic decisions after the event occurrence.

Proteomic Profiling of Sugar Beet (Beta vulgaris Leaves during Rhizomania Compatible Interactions

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Kimberly M. Webb

2014-04-01

Full Text Available Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV, severely impacts sugar beet (Beta vulgaris production throughout the world, and is widely prevalent in most production regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with BNYVV, but as resistance breaking strains become more prevalent, effective disease control strategies will require the application of novel methods based on better understanding of disease susceptibility and symptom development. Herein, proteomic profiling was conducted on susceptible sugar beet, infected with two strains of BNYVV, to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugar beet leaf tissue infected with BNYVV, quantified, and analyzed by mass spectrometry. A total of 203 proteins were confidently identified, with a predominance of proteins associated with photosynthesis and energy, metabolism, and response to stimulus. Many proteins identified in this study are typically associated with systemic acquired resistance and general plant defense responses. These results expand on relatively limited proteomic data available for sugar beet and provide the ground work for additional studies focused on understanding the interaction of BNYVV with sugar beet.

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.

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

Proteins in olive fruit and oil.

Science.gov (United States)

Montealegre, Cristina; Esteve, Clara; García, Maria Concepción; García-Ruiz, Carmen; Marina, Maria Luisa

2014-01-01

This paper is a comprehensive review grouping the information on the extraction, characterization, and quantitation of olive and olive oil proteins and providing a practical guide about these proteins. Most characterized olive proteins are located in the fruit, mainly in the seed, where different oleosins and storage proteins have been found. Unlike the seed, the olive pulp contains a lower protein content having been described a polypeptide of 4.6 kDa and a thaumain-like protein. Other important proteins studied in olive fruits have been enzymes which could play important roles in olives characteristics. Part of these proteins is transferred from the fruit to the oil during the manufacturing process of olive oil. In fact, the same polypeptide of 4.6 kDa found in the pulp has been described in the olive oil and, additionally, the presence of other proteins and enzymes have also been described. Protein profiles have recently been proposed as an interesting strategy for the varietal classification of olive fruits and oils. Nevertheless, there is still a lot of knowledge without being explored requiring new studies focused on the determination and characterization of these proteins.

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

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.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

International Nuclear Information System (INIS)

Hegele, Joerg; Buetler, Timo; Delatour, Thierry

2008-01-01

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N ε -fructoselysine (FL), N ε -carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lys free ) and 81.5 ± 87.8 nmol Pyr μmol -1 Lys free -1 vs. 3.72 ± 1.29 nmol FL μmol -1 Lys free -1 . In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol -1 of protein-bound Lys (Lys p-b ), 0.04 ± 0.03 nmol CML μmol -1 Lys p-b -1 and 0.06 ± 0.02 nmol Pyr μmol -1 Lys p-b -1 . It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products

Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

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Florian Heinke

2012-01-01

Full Text Available Diabetes insipidus (DI is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000–30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI. NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems.

Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

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Erhui Xiong

Full Text Available The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1 is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize and dicot (soybean and pea seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.

PanCoreGen – profiling, detecting, annotating protein-coding genes in microbial genomes

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Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V.

2015-01-01

A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen – a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars – Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. PMID:26456591

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.

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

International Nuclear Information System (INIS)

Herbette, L.; Blasie, J.K.

1984-01-01

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

Classification of protein profiles using fuzzy clustering techniques

DEFF Research Database (Denmark)

Karemore, Gopal; Mullick, Jhinuk B.; Sujatha, R.

2010-01-01

 Present  study  has  brought  out  a  comparison  of PCA  and  fuzzy  clustering  techniques  in  classifying  protein profiles  (chromatogram)  of  homogenates  of  different  tissue origins:  Ovarian,  Cervix,  Oral  cancers,  which  were  acquired using HPLC–LIF (High Performance Liquid...... Chromatography- Laser   Induced   Fluorescence)   method   developed   in   our laboratory. Study includes 11 chromatogram spectra each from oral,  cervical,  ovarian  cancers  as  well  as  healthy  volunteers. Generally  multivariate  analysis  like  PCA  demands  clear  data that   is   devoid   of   day......   PCA   mapping   in   classifying   various cancers from healthy spectra with classification rate up to 95 % from  60%.  Methods  are  validated  using  various  clustering indexes   and   shows   promising   improvement   in   developing optical pathology like HPLC-LIF for early detection of various...

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

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Vagner T. Paes de Oliveira

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

Changes in protein composition and protein phosphorylation during ...

African Journals Online (AJOL)

Changes in protein profiles and protein phosphorylation were studied in various stages of germinating somatic and zygotic embryos. Many proteins, which were expressed in cotyledonary stage somatic embryos, were also present in the zygotic embryos obtained from mature dry seed. The intensity of 22 kDa protein was ...

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.

Investigating homology between proteins using energetic profiles.

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Wrabl, James O; Hilser, Vincent J

2010-03-26

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

Investigating homology between proteins using energetic profiles.

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James O Wrabl

2010-03-01

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

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

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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.

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

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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.

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...

Identification of Protein Pupylation Sites Using Bi-Profile Bayes Feature Extraction and Ensemble Learning

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Xiaowei Zhao

2013-01-01

Full Text Available Pupylation, one of the most important posttranslational modifications of proteins, typically takes place when prokaryotic ubiquitin-like protein (Pup is attached to specific lysine residues on a target protein. Identification of pupylation substrates and their corresponding sites will facilitate the understanding of the molecular mechanism of pupylation. Comparing with the labor-intensive and time-consuming experiment approaches, computational prediction of pupylation sites is much desirable for their convenience and fast speed. In this study, a new bioinformatics tool named EnsemblePup was developed that used an ensemble of support vector machine classifiers to predict pupylation sites. The highlight of EnsemblePup was to utilize the Bi-profile Bayes feature extraction as the encoding scheme. The performance of EnsemblePup was measured with a sensitivity of 79.49%, a specificity of 82.35%, an accuracy of 85.43%, and a Matthews correlation coefficient of 0.617 using the 5-fold cross validation on the training dataset. When compared with other existing methods on a benchmark dataset, the EnsemblePup provided better predictive performance, with a sensitivity of 80.00%, a specificity of 83.33%, an accuracy of 82.00%, and a Matthews correlation coefficient of 0.629. The experimental results suggested that EnsemblePup presented here might be useful to identify and annotate potential pupylation sites in proteins of interest. A web server for predicting pupylation sites was developed.

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

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Yinliang Yang

2013-10-01

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

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.

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.

Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses, Hepatic Gene Expression, and Stress Protein Profiling.

Science.gov (United States)

Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

2016-02-01

In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na(+) -K(+) -ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation-related genes expression and stress-related proteins profiling in fish liver. Oxidative stress indices and Na(+) -K(+) -ATPase showed tissues-specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF-I axis and ion-regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose-dependent manner under TBT stress. In short, TBT-induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long-term experiments at lower concentrations will be necessary in the future. © 2015 Wiley Periodicals, Inc.

Magnetic field action on outdoor and indoor cultures of Spirulina: Evaluation of growth, medium consumption and protein profile.

Science.gov (United States)

Deamici, Kricelle Mosquera; Santos, Lucielen Oliveira; Costa, Jorge Alberto Vieira

2018-02-01

This study aimed at evaluating whether a magnetic field (MF) affects the growth of Spirulina sp. when applied to it at different exposure times in indoor and outdoor culture systems. The effects of MF on chlorophyll content, medium consumption and protein profile were also investigated. In raceway tanks, a 25 mT MF was applied for 24 h or for 1 h d -1 . MF for 24 h to outdoor assays increased biomass concentration and chlorophyll-a content besides altering the protein profile. Outdoor Spirulina growth was higher (∼3.65 g L -1 ) than the growth found in indoor assays (∼1.80 g L -1 ), while nitrogen and phosphorus consumption was not enhanced by the application of MF. This is the first study that investigated the influence of MF on outdoor microalga assays, and the results showed that MF affected the metabolism of Spirulina cultured in raceways, especially when it was grown outdoors in uncontrolled environmental conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential Protein Expression Profiles in Glaucomatous Trabecular Meshwork: An Evaluation Study on a Small Primary Open Angle Glaucoma Population.

Science.gov (United States)

Micera, Alessandra; Quaranta, Luciano; Esposito, Graziana; Floriani, Irene; Pocobelli, Augusto; Saccà, Sergio Claudio; Riva, Ivano; Manni, Gianluca; Oddone, Francesco

2016-02-01

Primary open angle glaucoma (POAG) is a progressive optic neuropathy characterized by impaired aqueous outflow and extensive remodeling in the trabecular meshwork (TM). The aim of this study was to characterize and compare the expression patterns of selected proteins belonging to the tissue remodeling, inflammation and growth factor pathways in ex vivo glaucomatous and post-mortem TMs using protein-array analysis. TM specimens were collected from 63 white subjects, including 40 patients with glaucoma and 23 controls. Forty POAG TMs were collected at the time of surgery and 23 post-mortem specimens were from non-glaucomatous donor sclerocorneal tissues. Protein profiles were evaluated using a chip-based array consisting of 60 literature-selected antibodies. A different expression of some factors was observed in POAG TMs with respect to post-mortem specimens, either in abundance (interleukin [IL]10, IL6, IL5, IL7, IL12, IL3, macrophage inflammatory protein [MIP]1δ/α, vascular endothelial growth factor [VEGF], transforming growth factor beta 1 [TGFβ1], soluble tumor necrosis factor receptor I [sTNFRI]) or in scarcity (IL16, IL18, intercellular adhesion molecule 3 [ICAM3], matrix metalloproteinase-7 [MMP7], tissue inhibitor of metalloproteinase 1 [TIMP1]). MMP2, MMP7, TGFβ1, and VEGF expressions were confirmed by Western blot, zymography, and polymerase chain reaction. No difference in protein profile expression was detected between glaucomatous subtypes. The analysis of this small TM population highlighted some proteins linked to POAG, some previously reported and others of new detection (IL7, MIPs, sTNFαRI). A larger POAG population is required to select promising disease-associated biomarker candidates. This study was partially supported by the Fondazione Roma, the Italian Ministry of Health and the "National 5xMille 2010 tax donation to IRCCS-G.B. Bietti Foundation".

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

DEFF Research Database (Denmark)

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

2009-01-01

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

Shave-off depth profiling: Depth profiling with an absolute depth scale

International Nuclear Information System (INIS)

Nojima, M.; Maekawa, A.; Yamamoto, T.; Tomiyasu, B.; Sakamoto, T.; Owari, M.; Nihei, Y.

2006-01-01

Shave-off depth profiling provides profiling with an absolute depth scale. This method uses a focused ion beam (FIB) micro-machining process to provide the depth profile. We show that the shave-off depth profile of a particle reflected the spherical shape of the sample and signal intensities had no relationship to the depth. Through the introduction of FIB micro-sampling, the shave-off depth profiling of a dynamic random access memory (DRAM) tip was carried out. The shave-off profile agreed with a blue print from the manufacturing process. Finally, shave-off depth profiling is discussed with respect to resolutions and future directions

Long-trace profiler for neutron focusing mirrors

International Nuclear Information System (INIS)

Puzyrev, Yevgeniy S.; Ice, Gene E.; Takacs, Peter Z.

2009-01-01

A long-trace profiler (LTP) optimized for measuring the shape of large neutron supermirrors has been designed and built. This LTP can measure 1.6 m long mirrors in both vertically and horizontally deflecting geometries, which is essential to achieve best performance from bendable mirrors. The LTP suppresses the influence of angular deviations of the linear-stage carriage during translation with a pentaprism and a cylindrical lens. The stationary optical head and the carriage-mounted pentaprism are precisely aligned to rotate about the optical axis between the two components. This feature allows measurements to be made on mirrors mounted vertically, horizontally or at any angle in between. The LTP software allows for rapid optimization of parameters for dynamically bent elliptical mirrors. Here we describe the motivation for the LTP, the design, and a first application of the LTP to study the effect of gravity on a bent microfocusing neutron supermirror.

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

Directory of Open Access Journals (Sweden)

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.

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

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Sasisekhar Bennuru

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

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

DEFF Research Database (Denmark)

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....

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

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

Science.gov (United States)

Wüst, Johannes; Pischetsrieder, Monika

2016-06-15

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

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

Science.gov (United States)

Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

2018-05-23

Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Protein Profiles Reveal Diverse Responsive Signaling Pathways in Kernels of Two Maize Inbred Lines with Contrasting Drought Sensitivity

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Liming Yang

2014-10-01

Full Text Available Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964 with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP, and protein profiles were investigated in developing kernels (35 DAP using iTRAQ (isobaric tags for relative and absolute quantitation. Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Energy Technology Data Exchange (ETDEWEB)

Hegele, Joerg [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)], E-mail: joerg.hegele@rdls.nestle.com; Buetler, Timo; Delatour, Thierry [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

2008-06-09

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N{sup {epsilon}}-fructoselysine (FL), N{sup {epsilon}}-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 {+-} 3.81 nmol CML per {mu}mol of free Lys (Lys{sub free}) and 81.5 {+-} 87.8 nmol Pyr {mu}mol{sup -1} Lys{sub free}{sup -1} vs. 3.72 {+-} 1.29 nmol FL {mu}mol{sup -1} Lys{sub free}{sup -1}. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 {+-} 0.08 nmol FL {mu}mol{sup -1} of protein-bound Lys (Lys{sub p-b}), 0.04 {+-} 0.03 nmol CML {mu}mol{sup -1} Lys{sub p-b}{sup -1} and 0.06 {+-} 0.02 nmol Pyr {mu}mol{sup -1} Lys{sub p-b}{sup -1}. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

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.

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.

Integrative structural modeling with small angle X-ray scattering profiles

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Schneidman-Duhovny Dina

2012-07-01

Full Text Available Abstract Recent technological advances enabled high-throughput collection of Small Angle X-ray Scattering (SAXS profiles of biological macromolecules. Thus, computational methods for integrating SAXS profiles into structural modeling are needed more than ever. Here, we review specifically the use of SAXS profiles for the structural modeling of proteins, nucleic acids, and their complexes. First, the approaches for computing theoretical SAXS profiles from structures are presented. Second, computational methods for predicting protein structures, dynamics of proteins in solution, and assembly structures are covered. Third, we discuss the use of SAXS profiles in integrative structure modeling approaches that depend simultaneously on several data types.

Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition

DEFF Research Database (Denmark)

Hansen, Mette; Lange, Marianne; Friis, Carsten

2007-01-01

Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need...... to be addressed. In the current study, a transcriptomic analysis of an antisense C-hordein line of barley was performed, using a grain-specific cDNA array. The C-hordein antisense line is characterized by marked changes in storage protein and amino acid profiles, while the seed weight is within the normal range...... and no external morphological irregularities were observed. The results of the transcriptome analysis showed excellent correlation with data on changes in the relative proportions of storage proteins and amino acid composition. The antisense line had a lower C-hordein level and down-regulated transcript encoding...

THE INFLUENCE OF AUTOLYSIS ON THE PROTEIN-PEPTIDE PROFILE OF Bos taurus AND Sus scrofa HEART AND AORTA TISSUES

Directory of Open Access Journals (Sweden)

I. M. Chernukha

2016-01-01

Full Text Available The article presents the results of autolytic processes impact on the protein-peptide profile of Bos taurus and Sus scrofa cardiac muscle and aorta. The results of tissue-specific protein identification are also presented as well as the effect of autolysis. Apolipoprotein A-1 involved in the formation of high-density lipoproteins, peroxiredoxin-1 involved in the suppression of oxidative stress, galectin-1 induced apoptosis of T-lymphocytes, as well as number of heat shock proteins with molecular weight less than 30 kDa were identified in Sus scrofa aorta tissue. It was discovered that functional proteins with molecular weight less than 30 kDa are retained during the freezing process, but destroyed under the action of autolytic enzymes. This work was supported by the Russian Science Foundation (project No. 16–16–10073.

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.

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

Science.gov (United States)

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

Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

Science.gov (United States)

Nguyen-Khuong, Terry; White, Melanie Y; Hung, Tzong-Tyng; Seeto, Shona; Thomas, Melissa L; Fitzgerald, Anna M; Martucci, Carlos E; Luk, Sharon; Pang, Shiu-Fu; Russell, Pamela J; Walsh, Bradley J

2009-04-01

Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

Effectiveness of Germination on Protein Hydrolysis as a Way To Reduce Adverse Reactions to Wheat.

Science.gov (United States)

Boukid, Fatma; Prandi, Barbara; Buhler, Sofie; Sforza, Stefano

2017-11-15

In this work, the aim is to study the effectiveness of germination on wheat protein degradation, with a specific focus on proteins involved in adverse reactions to wheat. The effects of 8 days of germination at 25 °C on the chemical composition and the protein profile were determined. Germination did not have a significant effect on starch, protein, lipid, and ash contents. General protein profile, as indicated by SDS-PAGE analysis, revealed that germination induced a relevant degradation in protein fraction. After in vitro gastrointestinal digestion, gluten peptides involved in celiac disease (CD) were identified and quantified using UPLC/ESI-MS technique. Also, CM3 protein, involved in baker's asthma and intestinal inflammation, was quantified by measuring a marker peptide. Statistical analysis underlined that germination and genotype had significant impact on the amount of both components. Regarding gluten peptides related to CD, germination enabled an average reduction of 47% in peptides eliciting adaptive immune response and 46% in peptides eliciting innate immune response. CM3 protein showed also a high average reduction (56%). Thus, this study suggests that germination might be a good bioalternative to provide a low "impact" raw ingredient for special wheat-based foodstuffs.

Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

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Liao Christopher CL

2010-08-01

Full Text Available Abstract Background Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS. Methods Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the 'GenePattern' suite of computational tools (Broad Institute, MIT, USA. Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR test statistic was used to identify and rank differentially expressed marker peaks. The k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, 'leave-one-out' cross-validation method. Results 73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour verses adjacent normal mucosa tissue (P ≤ 0.01, false discovery rate ≤ 0.05. Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100% sensitivity and specificity (95% confidence interval: 67.9-99.2%. Supervised prediction using 'leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours (P = P = P = 0.001; ROC error, 0.212. Conclusions Protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value in studies aimed at improved molecular

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.

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...

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.

Molecular spectroscopic features of protein in newly developed chickpea: Relationship with protein chemical profile and metabolism in the rumen and intestine of dairy cows

Science.gov (United States)

Sun, Baoli; Khan, Nazir Ahmad; Yu, Peiqiang

2018-05-01

The first aim of this study was to investigate the nutritional value of crude protein (CP) in CDC [Crop Development Centre (CDC), University of Saskatchewan] chickpea varieties (Frontier kabuli and Corinne desi) in comparison with a CDC barley variety in terms of: 1) CP chemical profile and subfractions; (2) in situ rumen degradation kinetics and intestinal digestibility of CP; 2) metabolizable protein (MP) supply to dairy cows; and (3) protein molecular structure characteristics using advanced molecular spectroscopy. The second aim was to quantify the relationship between protein molecular spectral characteristics and CP subfractions, in situ rumen CP degradation characteristics, intestinal digestibility of CP, and MP supply to dairy cows. Samples (n = 4) of each variety, from two consecutive years were analyzed. Chickpeas had higher (P content (21.71-22.11 vs 12.96% DM), with higher (P content, and any of the measured in situ degradation and molecular spectral characteristics of protein. The content of RUP was positively (r = 0.94, P content of CP (R2 = 0.91) D-fraction (R2 = 0.82), RDP (R2 = 0.77), RUP (R2 = 0.77), TDP (R2 = 0.98), MP (R2 = 0.80), and FMV (R2 = 0.80) can be predicted from amide II peak height. Despite extensive ruminal degradation, chickpea is a good source of MP for dairy cows, and molecular spectroscopy can be used to rapidly characterize feed protein molecular structures and predict their digestibility and nutritive value.

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.

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

Science.gov (United States)

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.

Careers in focus library and information science

CERN Document Server

2011-01-01

Careers in Focus: Library and Information Science, Second Edition profiles 19 careers for professionals interested in this field. Job profiles include:. -Acquisitions librarians. -Book conservators. -Children's librarians. -Corporate librarians. -Film and video librarians. -Law librarians. -Library assistants. -Library media specialists. -Medical librarians. -Research assistants.

Altered protein expression profiles in umbilical veins: insights into vascular dysfunctions of the children born after in vitro fertilization.

Science.gov (United States)

Gao, Qian; Pan, Hai-Tao; Lin, Xian-Hua; Zhang, Jun-Yu; Jiang, Ying; Tian, Shen; Chen, Lu-Ting; Liu, Miao-E; Xiong, Yi-Meng; Huang, He-Feng; Sheng, Jian-Zhong

2014-09-01

Cardiovascular dysfunction and remodeling have been found in some children conceived by in vitro fertilization (IVF). However, the underlying mechanisms remain unclear. In this study, the retrospective investigation showed that the blood pressure of IVF-conceived Chinese children was higher than that of naturally conceived (NC) children at ages 3-13 yr. We analyzed the expression profile of proteins in the umbilical veins of IVF and NC newborns by proteomic techniques. Using iTRAQ (isobaric tags for relative and absolute quantitation), 47 differentially expressed proteins (DEPs) were identified by feature selection in IVF umbilical veins compared with NC. Ingenuity Pathway Analysis, which is used to explore the signaling pathways of DEPs, revealed that these DEPs played important roles in vascular system development and carbon metabolism, implying that these DEPs might be potential candidates for further exploration of the mechanism(s) of vascular dysfunction in IVF children. We found that the serum estradiol (E₂) level in the cord blood of IVF newborns was significantly higher than that of NC newborns. High concentrations of E₂ induced alteration of lumican and vimentin expression in human umbilical vein endothelial cells, which was consistent with the proteomic results. These findings suggested that abnormal expression of proteins in umbilical veins might be related to the cardiovascular dysfunction and remodeling in IVF offspring. In conclusion, our data for the first time reveal the protein expression profile in blood vessels of IVF offspring and provide information for further mechanism study and evaluation of risks of cardiovascular abnormality in IVF children. © 2014 by the Society for the Study of Reproduction, Inc.

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.

Identification of biomarkers for radiation-induced acute intestinal symptoms (RIAISs) in cervical cancer patients by serum protein profiling

International Nuclear Information System (INIS)

Chai Yanlan; Wang Juan; Gao Ying

2015-01-01

Radiation-induced acute intestinal symptoms (RIAISs) are the most frequent complication of radiotherapy that causes great pain and limits the treatment efficacy. The aim of this study was to identify serum biomarkers of RIAISs in cervical cancer patients by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Serum samples were collected from 66 cervical cancer patients prior to pelvic radiotherapy. In our study, RIAISs occurred in 11 patients. An additional 11 patients without RIAISs were selected as controls, whose age, stage, histological type and treatment methods were matched to RIAISs patients. The 22 sera were subsequently analyzed by SELDI-TOF MS, and the resulting protein profiles were evaluated to identify biomarkers using appropriate bioinformatics tools. Comparing the protein profiles of serum samples from the RIAIS group and the control group, it was found that 22 protein peaks were significantly different (P < 0.05), and six of these peaks with mass-to-charge (m/z) ratios of 7514.9, 4603.94, 6887.41, 2769.21, 3839.72 and 4215.7 were successfully identified. A decision tree model of biomarkers was constructed based on three biomarkers (m/z 1270.88, 1503.23 and 7514.90), which separated RIAIS-affected patients from the control group with an accuracy of 81%. This study suggests that serum proteomic analysis by SELDI-TOF MS can identify cervical cancer patients that are susceptible to RIAISs prior to pelvic radiotherapy. (author)

Amino acid profile of metabolisable protein in lactating dairy cows is affected by dry matter concentration in grass-clover silage

DEFF Research Database (Denmark)

Johansen, Marianne; Lund, Peter; Weisbjerg, Martin Riis

2018-01-01

Our previous study showed that supply of metabolisable protein (MP) to lactating dairy cows increased with increasing dry matter (DM) concentration in grass-clover silage. The aim of this study was to examine how amino acid (AA) profile of MP was affected by silage DM concentration. Eight grass-c...

Surface glycosylation profiles of urine extracellular vesicles.

Directory of Open Access Journals (Sweden)

Jared Q Gerlach

Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

Directory of Open Access Journals (Sweden)

Nicholas T. Ingolia

2014-09-01

Full Text Available Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5′ UTRs and long noncoding RNAs (lncRNAs. Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs. Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

Clinical Trials of Precision Medicine through Molecular Profiling: Focus on Breast Cancer.

Science.gov (United States)

Zardavas, Dimitrios; Piccart-Gebhart, Martine

2015-01-01

High-throughput technologies of molecular profiling in cancer, such as gene-expression profiling and next-generation sequencing, are expanding our knowledge of the molecular landscapes of several cancer types. This increasing knowledge coupled with the development of several molecularly targeted agents hold the promise for personalized cancer medicine to be fully realized. Moreover, an expanding armamentarium of targeted agents has been approved for the treatment of specific molecular cancer subgroups in different diagnoses. According to this paradigm, treatment selection should be dictated by the specific molecular aberrations found in each patient's tumor. The classical clinical trials paradigm of patients' eligibility being based on clinicopathologic parameters is being abandoned, with current clinical trials enrolling patients on the basis of specific molecular aberrations. New, innovative trial designs have been generated to better tackle the multiple challenges induced by the increasing molecular fragmentation of cancer, namely: (1) longitudinal cohort studies with or without downstream trials, (2) studies assessing the clinical utility of molecular profiling, (3) master or umbrella trials, (4) basket trials, (5) N-of-1 trials, and (6) adaptive design trials. This article provides an overview of the challenges for clinical trials in the era of molecular profiling of cancer. Subsequently, innovative trial designs with respective examples and their potential to expedite efficient clinical development of targeted anticancer agents is discussed.

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/.

Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague–Dawley rats

Science.gov (United States)

Heden, Timothy D.; Morris, E. Matthew; Kearney, Monica L.; Liu, Tzu-Wen; Park, Young-min; Kanaley, Jill A.; Thyfault, John P.

2015-01-01

The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague–Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ~27% lower in HF- than in LF-fed animals (p < 0.05); the protein expression of all other molecules was similar in the 2 diets. The fed-state hepatic TAG secretion rate was ~39% lower (p < 0.05) in HF- (4.62 ± 0.18 mmol·h−1) than in LF- (7.60 ± 0.57 mmol·h−1) fed animals. Hepatic TAG content was ~2-fold higher (p < 0.05) in HF- (1.07 ± 0.15 nmol·g−1 tissue) than in LF- (0.50 ± 0.16 nmol·g−1 tissue) fed animals. In addition, the fatty acid profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression. PMID:24669989

Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague-Dawley rats.

Science.gov (United States)

Heden, Timothy D; Morris, E Matthew; Kearney, Monica L; Liu, Tzu-Wen; Park, Young-Min; Kanaley, Jill A; Thyfault, John P

2014-04-01

The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague-Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ∼27% lower in HF- than in LF-fed animals (p < 0.05); the protein expression of all other molecules was similar in the 2 diets. The fed-state hepatic TAG secretion rate was ∼39% lower (p < 0.05) in HF- (4.62 ± 0.18 mmol·h(-1)) than in LF- (7.60 ± 0.57 mmol·h(-1)) fed animals. Hepatic TAG content was ∼2-fold higher (p < 0.05) in HF- (1.07 ± 0.15 nmol·g(-1) tissue) than in LF- (0.50 ± 0.16 nmol·g(-1) tissue) fed animals. In addition, the fatty acid profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression.

Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

Directory of Open Access Journals (Sweden)

D Malfacini

Full Text Available Nociceptin/orphanin FQ (N/OFQ controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP. Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells. In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

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.

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.

Determination of the cesium-137 concentration profile in the main contamination focus of the radiological accident at Goiania-GO, Brazil

International Nuclear Information System (INIS)

Rocca, H.C.; Aoki, P.E.; Enokihara, C.T.; Rostelato, M.E.C.M.; Lepki, V.; Bambalas, E.

1988-01-01

The purpose of this paper is to describe the method used to determinate cesium-137 concentration profiles measured in function of depth and applied in seven areas considered as the main contamination focus. Since november 14 th to december 17 th 1987, 125 soil drillings were made and a total of 740 soil samples were prepared. Obtained data allowed to calculate the soil volume to be removed from contaminate areas. It was verified that after remotions the remaining activity was approximately 0,89 Ci. (author) [pt

Protein profiling of cerebrospinal fluid

DEFF Research Database (Denmark)

Simonsen, Anja H

2012-01-01

The cerebrospinal fluid (CSF) perfuses the brain and spinal cord. CSF contains proteins and peptides important for brain physiology and potentially also relevant for brain pathology. Hence, CSF is the perfect source to search for new biomarkers to improve diagnosis of neurological diseases as well...

New Technology-Large-Area Three- Dimensional Surface Profiling Using Only Focused Air-Coupled Ultrasound-Given 1999 R&D 100 Award

Science.gov (United States)

Roth, Don J.; Kautz, Harold E.; Abel, Phillip B.; Whalen, Mike F.; Hendricks, J. Lynne; Bodis, James R.

2000-01-01

Surface topography, which significantly affects the performance of many industrial components, is normally measured with diamond-tip profilometry over small areas or with optical scattering methods over larger areas. To develop air-coupled surface profilometry, the NASA Glenn Research Center at Lewis Field initiated a Space Act Agreement with Sonix, Inc., through two Glenn programs, the Advanced High Temperature Engine Materials Program (HITEMP) and COMMTECH. The work resulted in quantitative surface topography profiles obtained using only high-frequency, focused ultrasonic pulses in air. The method is nondestructive, noninvasive, and noncontact, and it does not require light-reflective surfaces. Air surface profiling may be desirable when diamond-tip or laserbased methods are impractical, such as over large areas, when a significant depth range is required, or for curved surfaces. When the configuration is optimized, the method is reasonably rapid and all the quantitative analysis facilities are online, including two- and three-dimensional visualization, extreme value filtering (for faulty data), and leveling.

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

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells.

Science.gov (United States)

Nyman, Tuula A; Lorey, Martina B; Cypryk, Wojciech; Matikainen, Sampsa

2017-05-01

The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses. Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses. Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.

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

Directory of Open Access Journals (Sweden)

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

Proteomic profiling of the rat hypothalamus

Directory of Open Access Journals (Sweden)

Pedroso Amanda P

2012-04-01

Full Text Available Abstract Background The hypothalamus plays a pivotal role in numerous mechanisms highly relevant to the maintenance of body homeostasis, such as the control of food intake and energy expenditure. Impairment of these mechanisms has been associated with the metabolic disturbances involved in the pathogenesis of obesity. Since rodent species constitute important models for metabolism studies and the rat hypothalamus is poorly characterized by proteomic strategies, we performed experiments aimed at constructing a two-dimensional gel electrophoresis (2-DE profile of rat hypothalamus proteins. Results As a first step, we established the best conditions for tissue collection and protein extraction, quantification and separation. The extraction buffer composition selected for proteome characterization of rat hypothalamus was urea 7 M, thiourea 2 M, CHAPS 4%, Triton X-100 0.5%, followed by a precipitation step with chloroform/methanol. Two-dimensional (2-D gels of hypothalamic extracts from four-month-old rats were analyzed; the protein spots were digested and identified by using tandem mass spectrometry and database query using the protein search engine MASCOT. Eighty-six hypothalamic proteins were identified, the majority of which were classified as participating in metabolic processes, consistent with the finding of a large number of proteins with catalytic activity. Genes encoding proteins identified in this study have been related to obesity development. Conclusion The present results indicate that the 2-DE technique will be useful for nutritional studies focusing on hypothalamic proteins. The data presented herein will serve as a reference database for studies testing the effects of dietary manipulations on hypothalamic proteome. We trust that these experiments will lead to important knowledge on protein targets of nutritional variables potentially able to affect the complex central nervous system control of energy homeostasis.

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

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Walderik W. Zomerman

2018-03-01

Full Text Available Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog, group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteome

Phylogenetic profiles of all membrane transport proteins of the malaria parasite highlight new drug targets

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January Weiner 3rd

2016-08-01

Full Text Available In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date.

A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins

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Mizuguchi Kenji

2008-09-01

Full Text Available Abstract Background Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. Results A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. Conclusion The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the α-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in

Cardiovascular and metabolic risk profile and acylation-stimulating protein levels in children with Prader-Willi syndrome and effects of growth hormone treatment

NARCIS (Netherlands)

R.F.A. de Lind van Wijngaarden (Roderick); K. Cianflone (Katherine); Y. Gao; R.W.J. Leunissen (Ralph); A.C.S. Hokken-Koelega (Anita)

2010-01-01

textabstractContext: Reports on the cardiovascular and metabolic risk profile in children with Prader-Willi syndrome (PWS) and the effects of GH treatment are scarce. Acylation-stimulating protein (ASP) stimulates glucose uptake and triglyceride storage in adipose tissue. Objectives: The aim was to

Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples

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Mark J Bailey

2013-01-01

Full Text Available Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4. The expression of TPM4 increased by 2-fold in Stage 2 before returning to "normal" levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage.

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

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Amar B. T. Ghisaidoobe

2014-12-01

Full Text Available F resonance energy transfer (FRET occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (\\(\\uplambda_{\\textsc{ex}}\\sim\\ nm, \\(\\uplambda_{\\textsc{em}}\\sim\\ 350 nm, in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the proteinlocal environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic F resonance energy transfer (iFRET, a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins.

Effect of canning on color, protein and phenolic profile of grains from kidney bean, field pea and chickpea.

Science.gov (United States)

Parmar, Naincy; Singh, Narpinder; Kaur, Amritpal; Virdi, Amardeep Singh; Thakur, Sheetal

2016-11-01

The aim of the present study was to evaluate the effect of canning on color, protein and phenolic profile of grains of kidney bean, field pea and chickpea varieties/accession. Color of grains of different pulses was enhanced after canning. Grains L* (lightness) decreased while a* (redness to yellowness) and b* (greenness to blueness) increased after canning in all the pulses. Protein profiling of grains of different pulses after canning revealed that kidney bean and chickpea, respectively, had the least and the most thermally susceptible polypeptides. Kidney bean and chickpea showed higher Percentage washed drained weight (PWDW) than field pea. Pulse with more grain hardness and PWDW showed higher degree of grain splitting during canning. Grain splitting was also higher in dark colored accessions/varieties as compared to the light colored. Ferulic acid was the most predominant compound present in raw grains of different pulses. Raw kidney bean grains showed higher accumulation of catechin, chlorogenic, protocatechuic acid, p-coumaric acid and ferulic acid than those of chickpea and field pea. Canning caused reduction in all the phenolic compounds except gallic acid and most prominent effect of canning on protocatechuic acid, chlorogenic and ferulic acid was observed. Copyright © 2016. Published by Elsevier Ltd.

Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis

Science.gov (United States)

Wang, Wei; Lu, Joann J.; Gu, Congying; Zhou, Lei; Liu, Shaorong

2013-01-01

In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl – polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl – polyacrylamide gel electrophoresis (SDS-PAGE, the 2nd-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

Country profile: Hungary

Energy Technology Data Exchange (ETDEWEB)

1991-09-01

Country Profile: Hungary has been prepared as a background document for use by US Government agencies and US businesses interested in becoming involved with the new democracies of Eastern Europe as they pursue sustainable economic development. The focus of the Profile is on energy and highlights information on Hungary`s energy supply, demand, and utilization. It identifies patterns of energy usage in the important economic sectors, especially industry, and provides a preliminary assessment for opportunities to improve efficiencies in energy production, distribution and use by introducing more efficient technologies. The use of more efficient technologies would have the added benefit of reducing the environmental impact which, although is not the focus of the report, is an issue that effects energy choices. The Profile also presents considerable economic information, primarily in the context of how economic restructuring may affect energy supply, demand, and the introduction of more efficient technologies.

Country profile: Hungary

Energy Technology Data Exchange (ETDEWEB)

1991-09-01

Country Profile: Hungary has been prepared as a background document for use by US Government agencies and US businesses interested in becoming involved with the new democracies of Eastern Europe as they pursue sustainable economic development. The focus of the Profile is on energy and highlights information on Hungary's energy supply, demand, and utilization. It identifies patterns of energy usage in the important economic sectors, especially industry, and provides a preliminary assessment for opportunities to improve efficiencies in energy production, distribution and use by introducing more efficient technologies. The use of more efficient technologies would have the added benefit of reducing the environmental impact which, although is not the focus of the report, is an issue that effects energy choices. The Profile also presents considerable economic information, primarily in the context of how economic restructuring may affect energy supply, demand, and the introduction of more efficient technologies.

Utilization of soya protein as an alternative protein source in ...

African Journals Online (AJOL)

STORAGESEVER

2009-01-05

Jan 5, 2009 ... For carcass trait, ash, crude fat, and energy varied significantly with soya protein ... high-protein content, relatively well-balanced amino acid profile ..... and organoleptic quality of flesh of brook char (Salvelinus fontinalis).

Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus.

Science.gov (United States)

Golo, Patrícia Silva; Dos Santos, Alessa Siqueira de Oliveira; Monteiro, Caio Marcio Oliveira; Perinotto, Wendell Marcelo de Souza; Quinelato, Simone; Camargo, Mariana Guedes; de Sá, Fillipe Araujo; Angelo, Isabele da Costa; Martins, Marta Fonseca; Prata, Marcia Cristina de Azevedo; Bittencourt, Vânia Rita Elias Pinheiro

2016-09-01

In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.

A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

Science.gov (United States)

Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

2016-08-05

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins.

Science.gov (United States)

Babcock, Joseph J; Li, Min

2014-01-01

The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided.

Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs

DEFF Research Database (Denmark)

Rasouli, Zahra; Valverde Pérez, Borja; D'Este, Martina

2018-01-01

wastewater and upcycle them to feed grade single cell protein. Results demonstrated that both algae and bacteria could remove or assimilate most of the organic carbon present in the wastewater (~95% removal for monocultures and 91% for the algal-bacterial consortium). However, their growth stopped before......, for all cultures the protein content (45% of dry weight, DW, for methanotrophs; 52.5% of DW for algae; and 27.6% of DW for consortium) and amino acid profile was suitable for substitution of conventional protein sources. Further research should focus on increasing productivity of biomass grown...

Eimeria Species and Genetic Background Influence the Serum Protein Profile of Broilers with Coccidiosis

Science.gov (United States)

Gilbert, Elizabeth R.; Cox, Chasity M.; Williams, Patricia M.; McElroy, Audrey P.; Dalloul, Rami A.; Ray, W. Keith; Barri, Adriana; Emmerson, Derek A.; Wong, Eric A.; Webb, Kenneth E.

2011-01-01

Background Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. Methodology/Principal Findings Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at PEimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods. PMID:21297942

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

Science.gov (United States)

Bhargava, Maneesh

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

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

International Nuclear Information System (INIS)

Berber, I.; Onlu, H.

2012-01-01

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

Predicting protein-ATP binding sites from primary sequence through fusing bi-profile sampling of multi-view features

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Zhang Ya-Nan

2012-05-01

Full Text Available Abstract Background Adenosine-5′-triphosphate (ATP is one of multifunctional nucleotides and plays an important role in cell biology as a coenzyme interacting with proteins. Revealing the binding sites between protein and ATP is significantly important to understand the functionality of the proteins and the mechanisms of protein-ATP complex. Results In this paper, we propose a novel framework for predicting the proteins’ functional residues, through which they can bind with ATP molecules. The new prediction protocol is achieved by combination of sequence evolutional information and bi-profile sampling of multi-view sequential features and the sequence derived structural features. The hypothesis for this strategy is single-view feature can only represent partial target’s knowledge and multiple sources of descriptors can be complementary. Conclusions Prediction performances evaluated by both 5-fold and leave-one-out jackknife cross-validation tests on two benchmark datasets consisting of 168 and 227 non-homologous ATP binding proteins respectively demonstrate the efficacy of the proposed protocol. Our experimental results also reveal that the residue structural characteristics of real protein-ATP binding sites are significant different from those normal ones, for example the binding residues do not show high solvent accessibility propensities, and the bindings prefer to occur at the conjoint points between different secondary structure segments. Furthermore, results also show that performance is affected by the imbalanced training datasets by testing multiple ratios between positive and negative samples in the experiments. Increasing the dataset scale is also demonstrated useful for improving the prediction performances.

UV-C Adaptation of Shigella: Morphological, Outer Membrane Proteins, Secreted Proteins, and Lipopolysaccharides Effects.

Science.gov (United States)

Chourabi, Kalthoum; Campoy, Susana; Rodriguez, Jesus A; Kloula, Salma; Landoulsi, Ahmed; Chatti, Abdelwaheb

2017-11-01

Water UV disinfection remains extremely important, particularly in developing countries where drinking and reclaimed crop irrigation water may spread devastating infectious diseases. Enteric bacterial pathogens, among which Shigella, are possible contaminants of drinking and bathing water and foods. To study the effect of UV light on Shigella, four strains were exposed to different doses in a laboratory-made irradiation device, given that the ultraviolet radiation degree of inactivation is directly related to the UV dose applied to water. Our results showed that the UV-C rays are effective against all the tested Shigella strains. However, UV-C doses appeared as determinant factors for Shigella eradication. On the other hand, Shigella-survived strains changed their outer membrane protein profiles, secreted proteins, and lipopolysaccharides. Also, as shown by electron microscopy transmission, morphological alterations were manifested by an internal cytoplasm disorganized and membrane envelope breaks. Taken together, the focus of interest of our study is to know the adaptive mechanism of UV-C resistance of Shigella strains.

Protein Comparability Assessments and Potential Applicability of High Throughput Biophysical Methods and Data Visualization Tools to Compare Physical Stability Profiles

Directory of Open Access Journals (Sweden)

Mohammad A. Alsenaidy

2014-03-01

Full Text Available In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs, radar charts, and comparative signature diagrams (CSDs for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1 mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF. Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress. Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles.

Science.gov (United States)

Alsenaidy, Mohammad A; Jain, Nishant K; Kim, Jae H; Middaugh, C Russell; Volkin, David B

2014-01-01

In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

Replacing dietary nonessential amino acids with ammonia nitrogen does not alter amino acid profile of deposited protein in the carcass of growing pigs fed a diet deficient in nonessential amino acid nitrogen.

Science.gov (United States)

Mansilla, W D; Htoo, J K; de Lange, C F M

2017-10-01

Amino acid usage for protein retention, and, consequently, the AA profile of retained protein, is the main factor for determining AA requirements in growing animals. The objective of the present study was to determine the effect of supplementing ammonia N on whole-body N retention and the AA profile of retained protein in growing pigs fed a diet deficient in nonessential AA (NEAA) N. In total, 48 barrows with a mean initial BW of 13.6 kg (SD 0.7) were used. At the beginning of the study, 8 pigs were euthanized for determination of initial protein mass. The remaining animals were individually housed and fed 1 of 5 dietary treatments. A common basal diet (95% of experimental diets) was formulated to meet the requirements for all essential AA (EAA) but to be deficient in NEAA N (CP = 8.01%). The basal diet was supplemented (5%) with cornstarch (negative control) or 2 N sources (ammonia or NEAA) at 2 levels each to supply 1.35 or 2.70% extra CP. The final standardized ileal digestible (SID) NEAA content in the high-NEAA-supplemented diet (positive control) was based on the NEAA profile of whole-body protein of 20-kg pigs, and it was expected to reduce the endogenous synthesis of NEAA. Pigs were fed at 3.0 times maintenance energy requirements for ME in 3 equal meals daily. At the end of a 3-wk period, pigs were euthanized and the carcass and visceral organs were weighed, frozen, and ground for determination of protein mass. From pigs in the initial, negative control, high-ammonia, and high-NEAA groups, AA contents in the carcass and pooled visceral organs were analyzed to determine the total and deposited protein AA profile, dietary EAA efficiencies, and minimal de novo synthesis of NEAA. Carcass weight and whole-body N retention linearly increased ( 0.10) between N sources, but Cys content increased ( ammonia in visceral organ protein and deposited protein. The dietary SID EAA efficiency for increasing EAA deposition in whole-body protein increased ( 0.10) between N

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

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Adriana FIORINI

2016-01-01

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

Phylo_dCor: distance correlation as a novel metric for phylogenetic profiling.

Science.gov (United States)

Sferra, Gabriella; Fratini, Federica; Ponzi, Marta; Pizzi, Elisabetta

2017-09-05

Elaboration of powerful methods to predict functional and/or physical protein-protein interactions from genome sequence is one of the main tasks in the post-genomic era. Phylogenetic profiling allows the prediction of protein-protein interactions at a whole genome level in both Prokaryotes and Eukaryotes. For this reason it is considered one of the most promising methods. Here, we propose an improvement of phylogenetic profiling that enables handling of large genomic datasets and infer global protein-protein interactions. This method uses the distance correlation as a new measure of phylogenetic profile similarity. We constructed robust reference sets and developed Phylo-dCor, a parallelized version of the algorithm for calculating the distance correlation that makes it applicable to large genomic data. Using Saccharomyces cerevisiae and Escherichia coli genome datasets, we showed that Phylo-dCor outperforms phylogenetic profiling methods previously described based on the mutual information and Pearson's correlation as measures of profile similarity. In this work, we constructed and assessed robust reference sets and propose the distance correlation as a measure for comparing phylogenetic profiles. To make it applicable to large genomic data, we developed Phylo-dCor, a parallelized version of the algorithm for calculating the distance correlation. Two R scripts that can be run on a wide range of machines are available upon request.

Ingredient classification according to the digestible amino acid profile: an exploratory analysis

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DE Faria Filho

2005-09-01

Full Text Available This study aimed: 1 to classify ingredients according to the digestible amino acid (AA profile; 2 to determine ingredients with AA profile closer to the ideal for broiler chickens; and 3 to compare digestible AA profiles from simulated diets with the ideal protein profile. The digestible AA levels of 30 ingredients were compiled from the literature and presented as percentages of lysine according to the ideal protein concept. Cluster and principal component analyses (exploratory analyses were used to compose and describe groups of ingredients according to AA profiles. Four ingredient groups were identified by cluster analysis, and the classification of the ingredients within each of these groups was obtained from a principal component analysis, showing 11 classes of ingredients with similar digestible AA profiles. The ingredients with AA profiles closer to the ideal protein were meat and bone meal 45, fish meal 60 and wheat germ meal, all of them constituting Class 1; the ingredients from the other classes gradually diverged from the ideal protein. Soybean meal, which is the main protein source for poultry, showed good AA balance since it was included in Class 3. On the contrary, corn, which is the main energy source in poultry diets, was classified in Class 8. Dietary AA profiles were improved when corn and/or soybean meal were partially or totally replaced in the simulations by ingredients with better AA balance.

Species identification of smoked and gravad fish products by sodium dodecylsulphate polyacrylamide gel electrophoresis, urea isoelectric focusing and native isoelectric focusing : a collaborative study

DEFF Research Database (Denmark)

Mackie, I.; Craig, A.; Etienne, M.

2000-01-01

A collaborative study on the use of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out by eight laborator......A collaborative study on the use of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out by eight...... laboratories. With SDS-PAGE, minor changes took place in the profiles of the processed salmonid species making it impossible or Very difficult to identify closely related species. With urea-IEF, there were fewer changes in the profiles due to processing and the system generally had greater species......-discriminating power for the processed salmonids than SDS-PAGE. The profiles of the eel species as obtained on SDS-PAGE or urea-IEF were not affected by smoking. Urea-IEF had greater species- discriminating power than SDS-PAGE for the eel species. Native IEF was useful in providing supplementary identification...

Depletion field focusing in semiconductors

NARCIS (Netherlands)

Prins, M.W.J.; Gelder, Van A.P.

1996-01-01

We calculate the three-dimensional depletion field profile in a semiconductor, for a planar semiconductor material with a spatially varying potential upon the surface, and for a tip-shaped semiconductor with a constant surface potential. The nonuniform electric field gives rise to focusing or

Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

International Nuclear Information System (INIS)

Liao, Christopher CL; Ward, Nicholas; Marsh, Simon; Arulampalam, Tan; Norton, John D

2010-01-01

Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS). Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the 'GenePattern' suite of computational tools (Broad Institute, MIT, USA). Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR) test statistic was used to identify and rank differentially expressed marker peaks. The k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, 'leave-one-out' cross-validation method. 73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour verses adjacent normal mucosa tissue (P ≤ 0.01, false discovery rate ≤ 0.05). Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100% sensitivity and specificity (95% confidence interval: 67.9-99.2%). Supervised prediction using 'leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours (P = < 0.001; receiver-operator characteristics - ROC - error, 0.171); disease recurrence was correctly predicted in 5/6 cases and disease-free survival (median follow-up time, 25 months) was correctly predicted in 22

Human Serum Protein-Bound iodine and Protein Fractions at ...

African Journals Online (AJOL)

Iodine profile of Nigerians at different ages in both sexes and in pregnant women, and under narcotic influence, such as alcoholism, cigarette smoking and marijuana addiction were studied. Their serum total protein, albumin and globulin concentrations were also determined. Results of the study showed that serum protein ...

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

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Marius Schild

2016-01-01

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

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

Energy Technology Data Exchange (ETDEWEB)

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

2016-01-29

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH₃) to nitrite (NO₂^₋) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH₄⁺-dependent O₂uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and

Swiftly moving focus points and forming shapes through the scattering media

Science.gov (United States)

Tran, Vinh; Sahoo, Sujit Kumar; Tang, Dongliang; Dang, Cuong

2018-02-01

Propagation of light through scattering media such as ground glass or biological tissue limits the quality and intensity of focusing point. Wave front shaping technique which uses spatial light modulator (SLM) devices to reshape the field profile of incoming light, is considered as one of the most effective and convenient methods. Advanced biomedical or manufacturing applications require drawing various contours or shapes quickly and precisely. However, creating each shape behind the scattering medium needs different phase profiles, which are time consuming to optimize or measure. Here, we demonstrate a technique to draw various shapes or contours behind the scattering medium by swiftly moving the focus point without any mechanical movements. Our technique relies on the existence of speckle correlation property in scattering media, also known as optical memory effect. In our procedure, we first modulate the phase-only SLM to create the focus point on the other side of scattering medium. Then, we digitally shift the preoptimized phase profile on the SLM and ramp it to tilt the beam accordingly. Now, the incoming beam with identical phase profile shines on the same scattering region at a tilted angle to regenerate the focus point at the desired position due to memory effect. Moreover, with linear combination of different field patterns, we can generate a single phase profile on SLM to produce two, three or more focus points simultaneously on the other side of a turbid medium. Our method could provide a useful tool for prominent applications such as opto-genetic excitation, minimally invasive laser surgery and other related fields.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

Science.gov (United States)

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

HMMEditor: a visual editing tool for profile hidden Markov model

Directory of Open Access Journals (Sweden)

Cheng Jianlin

2008-03-01

Full Text Available Abstract Background Profile Hidden Markov Model (HMM is a powerful statistical model to represent a family of DNA, RNA, and protein sequences. Profile HMM has been widely used in bioinformatics research such as sequence alignment, gene structure prediction, motif identification, protein structure prediction, and biological database search. However, few comprehensive, visual editing tools for profile HMM are publicly available. Results We develop a visual editor for profile Hidden Markov Models (HMMEditor. HMMEditor can visualize the profile HMM architecture, transition probabilities, and emission probabilities. Moreover, it provides functions to edit and save HMM and parameters. Furthermore, HMMEditor allows users to align a sequence against the profile HMM and to visualize the corresponding Viterbi path. Conclusion HMMEditor provides a set of unique functions to visualize and edit a profile HMM. It is a useful tool for biological sequence analysis and modeling. Both HMMEditor software and web service are freely available.

Analysis of post-operative changes in serum protein expression profiles from colorectal cancer patients by MALDI-TOF mass spectrometry: a pilot methodological study

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Marsh Simon

2010-04-01

Full Text Available Abstract Background Mass spectrometry-based protein expression profiling of blood sera can be used to discriminate colorectal cancer (CRC patients from unaffected individuals. In a pilot methodological study, we have evaluated the changes in protein expression profiles of sera from CRC patients that occur following surgery to establish the potential of this approach for monitoring post-surgical response and possible early prediction of disease recurrence. Methods In this initial pilot study, serum specimens from 11 cancer patients taken immediately prior to surgery and at approximately 6 weeks following surgery were analysed alongside 10 normal control sera by matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS. Using a two-sided t-test the top 20 ranked protein peaks that discriminate normal from pre-operative sera were identified. These were used to classify post-operative sera by hierarchical clustering analysis (Spearman's Rank correlation and, as an independent 'test' dataset, by k-nearest neighbour and weighted voting supervised learning algorithms. Results Hierarchical cluster analysis classified post-operative sera from all six early Dukes' stage (A and B patients as normal. The remaining five post-operative sera from more advanced Dukes' stages (C1 and C2 were classified as cancer. Analysis by supervised learning algorithms similarly grouped all advanced Dukes' stages as cancer, with four of the six post-operative sera from early Dukes' stages being classified as normal (P = 0.045; Fisher's exact test. Conclusions The results of this pilot methodological study illustrate the proof-of-concept of using protein expression profiling of post-surgical blood sera from individual patients to monitor disease course. Further validation on a larger patient cohort and using an independent post-operative sera dataset would be required to evaluate the potential clinical relevance of this approach. Prospective

A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction.

Science.gov (United States)

Li, Ying; Xu, Fei; Liu, Chao; Xu, Youzhi; Feng, Xiaojun; Liu, Bi-Feng

2013-08-21

Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

Globular and disordered-the non-identical twins in protein-protein interactions

DEFF Research Database (Denmark)

Teilum, Kaare; Olsen, Johan Gotthardt; Kragelund, Birthe Brandt

2015-01-01

as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those...... of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol(-1)....

Poly(lactic-co-glycolic acid) devices: Production and applications for sustained protein delivery.

Science.gov (United States)

Lee, Parker W; Pokorski, Jonathan K

2018-03-13

Injectable or implantable poly(lactic-co-glycolic acid) (PLGA) devices for the sustained delivery of proteins have been widely studied and utilized to overcome the necessity of repeated administrations for therapeutic proteins due to poor pharmacokinetic profiles of macromolecular therapies. These devices can come in the form of microparticles, implants, or patches depending on the disease state and route of administration. Furthermore, the release rate can be tuned from weeks to months by controlling the polymer composition, geometry of the device, or introducing additives during device fabrication. Slow-release devices have become a very powerful tool for modern medicine. Production of these devices has initially focused on emulsion-based methods, relying on phase separation to encapsulate proteins within polymeric microparticles. Process parameters and the effect of additives have been thoroughly researched to ensure protein stability during device manufacturing and to control the release profile. Continuous fluidic production methods have also been utilized to create protein-laden PLGA devices through spray drying and electrospray production. Thermal processing of PLGA with solid proteins is an emerging production method that allows for continuous, high-throughput manufacturing of PLGA/protein devices. Overall, polymeric materials for protein delivery remain an emerging field of research for the creation of single administration treatments for a wide variety of disease. This review describes, in detail, methods to make PLGA devices, comparing traditional emulsion-based methods to emerging methods to fabricate protein-laden devices. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Peptide-Based Structures. © 2018 Wiley Periodicals, Inc.

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.

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

Science.gov (United States)

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

2015-07-01

The study examined the effect of various processing methods- boiling, drying and roasting- on the in vitro and in vivo protein digestibility and free amino acid profiles of Terminalia catappa seed. Moisture and crude protein of the various samples were determined. In vitro protein digestibility was determined after pepsin digestion. For the in vivo experiment, defatted T. catappa based diet was fed to 3 weeks old Wistar rats for 4 weeks and compared with animals maintained on casein based and nitrogen- free diets. The biological value (BV), net protein utilisation (NPU) and protein efficiency ratio (PER) of the diets were determined. Free amino acid composition was carried out using thin layer chromatography. Moisture was highest in the boiled T. catappa seed (8.30 ± 0.00 %). The raw, roasted and dried seeds had 5.55 ± 0.07, 3.88 ± 0.22 and 3.75 ± 0.07 % respectively. Crude protein was 19.19, 18.89, 17.62 and 16.36 % in the dried, roasted, boiled and raw seeds respectively. Roasted T. catappa seed had the highest in vitro protein digestibility with 37.52 %, while the dried, boiled and raw samples had digestibility values of 27.57, 27.07 and 24.45 % respectively. All nine essential amino acids were present in T. catappa in high concentrations except methionine and tryptophan. Glutamate was present in the highest concentration. Also, free amino acids were higher in the processed seeds compared to the raw seed. Animals fed T. catappa diet compared favourably with the casein group, thus indicating that the protein is of good quality.

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

Directory of Open Access Journals (Sweden)

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

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins.

Science.gov (United States)

Mena, Ma Luz; Moreno-Gordaliza, Estefanía; Moraleja, Irene; Cañas, Benito; Gómez-Gómez, Ma Milagros

2011-03-04

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated. Copyright © 2011 Elsevier B.V. All rights reserved.

Genome-wide identification of VQ motif-containing proteins and their expression profiles under abiotic stresses in maize

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Weibin eSong

2016-01-01

Full Text Available VQ motif-containing proteins play crucial roles in abiotic stress responses in plants. Recent studies have shown that some VQ proteins physically interact with WRKY transcription factors to activate downstream genes. In the present study, we identified and characterized genes encoding VQ motif-containing proteins using the most recent version of the maize genome sequence. In total, 61VQ genes were identified. In a cluster analysis, these genes clustered into nine groups together with their homologous genes in rice and Arabidopsis. Most of the VQ genes (57 out of 61 numbers identified in maize were found to be single-copy genes. Analyses of RNA-seq data obtained using seedlings under long-term drought treatment showed that the expression levels of most ZmVQ genes (41 out of 61 members changed during the drought stress response. Quantitative real-time PCR analyses showed that most of the ZmVQ genes were responsive to NaCl treatment. Also, approximately half of the ZmVQ genes were co-expressed with ZmWRKY genes. The identification of these VQ genes in the maize genome and knowledge of their expression profiles under drought and osmotic stresses will provide a solid foundation for exploring their specific functions in the abiotic stress responses of maize.

Gene-metabolite profile integration to understand the cause of spaceflight induced immunodeficiency.

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Chakraborty, Nabarun; Cheema, Amrita; Gautam, Aarti; Donohue, Duncan; Hoke, Allison; Conley, Carolynn; Jett, Marti; Hammamieh, Rasha

2018-01-01

Spaceflight presents a spectrum of stresses very different from those associated with terrestrial conditions. Our previous study (BMC Genom. 15 : 659, 2014) integrated the expressions of mRNAs, microRNAs, and proteins and results indicated that microgravity induces an immunosuppressive state that can facilitate opportunistic pathogenic attack. However, the existing data are not sufficient for elucidating the molecular drivers of the given immunosuppressed state. To meet this knowledge gap, we focused on the metabolite profile of spaceflown human cells. Independent studies have attributed cellular energy deficiency as a major cause of compromised immunity of the host, and metabolites that are closely associated with energy production could be a robust signature of atypical energy fluctuation. Our protocol involved inoculation of human endothelial cells in cell culture modules in spaceflight and on the ground concurrently. Ten days later, the cells in space and on the ground were exposed to lipopolysaccharide (LPS), a ubiquitous membrane endotoxin of Gram-negative bacteria. Nucleic acids, proteins, and metabolites were collected 4 and 8 h post-LPS exposure. Untargeted profiling of metabolites was followed by targeted identification of amino acids and knowledge integration with gene expression profiles. Consistent with the past reports associating microgravity with increased energy expenditure, we identified several markers linked to energy deficiency, including various amino acids such as tryptophan, creatinine, dopamine, and glycine, and cofactors such as lactate and pyruvate. The present study revealed a molecular architecture linking energy metabolism and immunodeficiency in microgravity. The energy-deficient condition potentially cascaded into dysregulation of protein metabolism and impairment of host immunity. This project is limited by a small sample size. Although a strict statistical screening was carefully implemented, the present results further emphasize

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

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Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

2016-04-01

The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-)Sca-1(+) CD49f(+) Trop2(high)-phenotype) and human (Lin(-) CD49f(+) TROP2(high)) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+)/TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+)/CD24(+)/CD29(+)/CD44(+)/CD47(+)/CD49f(+)/CD104(+)/CD147(+)/CD326(+)/Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by

Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

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Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng; Liu Zhen

2009-01-01

Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 ± 0.83 x 10 -3 s -1 . The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

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Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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2013-01-01

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

ABC gene expression profiles have clinical importance and possibly form a new hallmark of cancer.

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Dvorak, Pavel; Pesta, Martin; Soucek, Pavel

2017-05-01

Adenosine triphosphate-binding cassette proteins constitute a large family of active transporters through extracellular and intracellular membranes. Increased drug efflux based on adenosine triphosphate-binding cassette protein activity is related to the development of cancer cell chemoresistance. Several articles have focused on adenosine triphosphate-binding cassette gene expression profiles (signatures), based on the expression of all 49 human adenosine triphosphate-binding cassette genes, in individual tumor types and reported connections to established clinicopathological features. The aim of this study was to test our theory about the existence of adenosine triphosphate-binding cassette gene expression profiles common to multiple types of tumors, which may modify tumor progression and provide clinically relevant information. Such general adenosine triphosphate-binding cassette profiles could constitute a new attribute of carcinogenesis. Our combined cohort consisted of tissues from 151 cancer patients-breast, colorectal, and pancreatic carcinomas. Standard protocols for RNA isolation and quantitative real-time polymerase chain reaction were followed. Gene expression data from individual tumor types as well as a merged tumor dataset were analyzed by bioinformatics tools. Several general adenosine triphosphate-binding cassette profiles, with differences in gene functions, were established and shown to have significant relations to clinicopathological features such as tumor size, histological grade, or clinical stage. Genes ABCC7, A3, A8, A12, and C8 prevailed among the most upregulated or downregulated ones. In conclusion, the results supported our theory about general adenosine triphosphate-binding cassette gene expression profiles and their importance for cancer on clinical as well as research levels. The presence of ABCC7 (official symbol CFTR) among the genes with key roles in the profiles supports the emerging evidence about its crucial role in various

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.

Study of the proteins in the defatted flour and protein concentrate of baru nuts (Dipteryx alata Vog

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Rita de Cássia Avellaneda Guimarães

2012-09-01

Full Text Available Baru (Dipteryx alata Vog. is an abundant legume in the Brazilian Savanna. Its nuts can be exploited sustainably using its protein and lipid fractions. This study aimed to analyze the proteins of the nuts present in the defatted flour and protein concentrate in terms of their functional properties, the profile of their fractions, and the in vitro digestibility. The flour was defatted with hexane and extracted at the pH of higher protein solubility to obtain the protein concentrate. The electrophoretic profile of the protein fractions was evaluated in SDS-PAGE gel. The functional properties of the proteins indicate the possibility of their use in various foods, like soybeans providing water absorption capacity, oil absorption capacity, emulsifying properties, and foamability. Globulins, followed by the albumins, are the major fractions of the flour and protein concentrate, respectively. Digestibility was greater for the concentrate than for the defatted flour.

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

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Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-01-01

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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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.

Evaluation of GO-based functional similarity measures using S. cerevisiae protein interaction and expression profile data

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Du LinFang

2008-11-01

Full Text Available Abstract Background Researchers interested in analysing the expression patterns of functionally related genes usually hope to improve the accuracy of their results beyond the boundaries of currently available experimental data. Gene ontology (GO data provides a novel way to measure the functional relationship between gene products. Many approaches have been reported for calculating the similarities between two GO terms, known as semantic similarities. However, biologists are more interested in the relationship between gene products than in the scores linking the GO terms. To highlight the relationships among genes, recent studies have focused on functional similarities. Results In this study, we evaluated five functional similarity methods using both protein-protein interaction (PPI and expression data of S. cerevisiae. The receiver operating characteristics (ROC and correlation coefficient analysis of these methods showed that the maximum method outperformed the other methods. Statistical comparison of multiple- and single-term annotated proteins in biological process ontology indicated that genes with multiple GO terms may be more reliable for separating true positives from noise. Conclusion This study demonstrated the reliability of current approaches that elevate the similarity of GO terms to the similarity of proteins. Suggestions for further improvements in functional similarity analysis are also provided.

Focused conformational sampling in proteins

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Bacci, Marco; Langini, Cassiano; Vymětal, Jiří; Caflisch, Amedeo; Vitalis, Andreas

2017-11-01

A detailed understanding of the conformational dynamics of biological molecules is difficult to obtain by experimental techniques due to resolution limitations in both time and space. Computer simulations avoid these in theory but are often too short to sample rare events reliably. Here we show that the progress index-guided sampling (PIGS) protocol can be used to enhance the sampling of rare events in selected parts of biomolecules without perturbing the remainder of the system. The method is very easy to use as it only requires as essential input a set of several features representing the parts of interest sufficiently. In this feature space, new states are discovered by spontaneous fluctuations alone and in unsupervised fashion. Because there are no energetic biases acting on phase space variables or projections thereof, the trajectories PIGS generates can be analyzed directly in the framework of transition networks. We demonstrate the possibility and usefulness of such focused explorations of biomolecules with two loops that are part of the binding sites of bromodomains, a family of epigenetic "reader" modules. This real-life application uncovers states that are structurally and kinetically far away from the initial crystallographic structures and are also metastable. Representative conformations are intended to be used in future high-throughput virtual screening campaigns.

Integration of miRNA and protein profiling reveals coordinated neuroadaptations in the alcohol-dependent mouse brain.

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Giorgio Gorini

Full Text Available The molecular mechanisms underlying alcohol dependence involve different neurochemical systems and are brain region-dependent. Chronic Intermittent Ethanol (CIE procedure, combined with a Two-Bottle Choice voluntary drinking paradigm, represents one of the best available animal models for alcohol dependence and relapse drinking. MicroRNAs, master regulators of the cellular transcriptome and proteome, can regulate their targets in a cooperative, combinatorial fashion, ensuring fine tuning and control over a large number of cellular functions. We analyzed cortex and midbrain microRNA expression levels using an integrative approach to combine and relate data to previous protein profiling from the same CIE-subjected samples, and examined the significance of the data in terms of relative contribution to alcohol consumption and dependence. MicroRNA levels were significantly altered in CIE-exposed dependent mice compared with their non-dependent controls. More importantly, our integrative analysis identified modules of coexpressed microRNAs that were highly correlated with CIE effects and predicted target genes encoding differentially expressed proteins. Coexpressed CIE-relevant proteins, in turn, were often negatively correlated with specific microRNA modules. Our results provide evidence that microRNA-orchestrated translational imbalances are driving the behavioral transition from alcohol consumption to dependence. This study represents the first attempt to combine ex vivo microRNA and protein expression on a global scale from the same mammalian brain samples. The integrative systems approach used here will improve our understanding of brain adaptive changes in response to drug abuse and suggests the potential therapeutic use of microRNAs as tools to prevent or compensate multiple neuroadaptations underlying addictive behavior.

High-Specificity Targeted Functional Profiling in Microbial Communities with ShortBRED.

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James Kaminski

2015-12-01

Full Text Available Profiling microbial community function from metagenomic sequencing data remains a computationally challenging problem. Mapping millions of DNA reads from such samples to reference protein databases requires long run-times, and short read lengths can result in spurious hits to unrelated proteins (loss of specificity. We developed ShortBRED (Short, Better Representative Extract Dataset to address these challenges, facilitating fast, accurate functional profiling of metagenomic samples. ShortBRED consists of two components: (i a method that reduces reference proteins of interest to short, highly representative amino acid sequences ("markers" and (ii a search step that maps reads to these markers to quantify the relative abundance of their associated proteins. After evaluating ShortBRED on synthetic data, we applied it to profile antibiotic resistance protein families in the gut microbiomes of individuals from the United States, China, Malawi, and Venezuela. Our results support antibiotic resistance as a core function in the human gut microbiome, with tetracycline-resistant ribosomal protection proteins and Class A beta-lactamases being the most widely distributed resistance mechanisms worldwide. ShortBRED markers are applicable to other homology-based search tasks, which we demonstrate here by identifying phylogenetic signatures of antibiotic resistance across more than 3,000 microbial isolate genomes. ShortBRED can be applied to profile a wide variety of protein families of interest; the software, source code, and documentation are available for download at http://huttenhower.sph.harvard.edu/shortbred.

Development of an activity-based probe for acyl-protein thioesterases

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Garland, Megan; Schulze, Christopher J.; Foe, Ian T.; van der Linden, Wouter A.; Child, Matthew A.

2018-01-01

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe. PMID:29364904

Protein and antigen profiles of Leptospira interrogans serovar Hardjo Perfil proteico e antigênico da Leptospira interrogans sorovariedade Hardjo

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Bárbara Nobre Lafetá

2009-12-01

Full Text Available The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.Estudou-se o perfil proteico da membrana externa da Leptospira interrogans sorovariedade Hardjo, amostra hardjoprajitno, associado à resposta imune natural de bovinos infectados. Foram utilizados Triton X114 para a extração das proteínas de membrana externa e acetona para precipitá-las. As proteínas extraídas foram analisadas por SDS-PAGE e western blot contra soro de coelhos hiperimunes e de bovinos naturalmente infectados. Em géis corados com nitrato de prata, 14 bandas proteicas foram identificadas, e quatro dessas bandas, com 22, 29, 47 e 63kDa, foram as mais proeminentes. Os western blots com soro hiperimune de coelho detectaram bandas correspondentes a proteínas com pesos moleculares de 35, 27, 24, 21, 17 e 14kDa, e bandas de 32 e 45kDa destacaram-se nos testes com soros de bovinos naturalmente infectados.

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

International Nuclear Information System (INIS)

Munir, T.A.; Afzal, M.N.

2010-01-01

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

Global proteomics profiling improves drug sensitivity prediction: results from a multi-omics, pan-cancer modeling approach.

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Ali, Mehreen; Khan, Suleiman A; Wennerberg, Krister; Aittokallio, Tero

2018-04-15

Proteomics profiling is increasingly being used for molecular stratification of cancer patients and cell-line panels. However, systematic assessment of the predictive power of large-scale proteomic technologies across various drug classes and cancer types is currently lacking. To that end, we carried out the first pan-cancer, multi-omics comparative analysis of the relative performance of two proteomic technologies, targeted reverse phase protein array (RPPA) and global mass spectrometry (MS), in terms of their accuracy for predicting the sensitivity of cancer cells to both cytotoxic chemotherapeutics and molecularly targeted anticancer compounds. Our results in two cell-line panels demonstrate how MS profiling improves drug response predictions beyond that of the RPPA or the other omics profiles when used alone. However, frequent missing MS data values complicate its use in predictive modeling and required additional filtering, such as focusing on completely measured or known oncoproteins, to obtain maximal predictive performance. Rather strikingly, the two proteomics profiles provided complementary predictive signal both for the cytotoxic and targeted compounds. Further, information about the cellular-abundance of primary target proteins was found critical for predicting the response of targeted compounds, although the non-target features also contributed significantly to the predictive power. The clinical relevance of the selected protein markers was confirmed in cancer patient data. These results provide novel insights into the relative performance and optimal use of the widely applied proteomic technologies, MS and RPPA, which should prove useful in translational applications, such as defining the best combination of omics technologies and marker panels for understanding and predicting drug sensitivities in cancer patients. Processed datasets, R as well as Matlab implementations of the methods are available at https://github.com/mehr-een/bemkl-rbps. mehreen

Comparative study of the protein profiles of Sunki mandarin and Rangpur lime plants in response to water deficit.

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Oliveira, Tahise M; da Silva, Fernanda R; Bonatto, Diego; Neves, Diana M; Morillon, Raphael; Maserti, Bianca E; Filho, Mauricio A Coelho; Costa, Marcio G C; Pirovani, Carlos P; Gesteira, Abelmon S

2015-03-03

Rootstocks play a major role in the tolerance of citrus plants to water deficit by controlling and adjusting the water supply to meet the transpiration demand of the shoots. Alterations in protein abundance in citrus roots are crucial for plant adaptation to water deficit. We performed two-dimensional electrophoresis (2-DE) separation followed by LC/MS/MS to assess the proteome responses of the roots of two citrus rootstocks, Rangpur lime (Citrus limonia Osbeck) and 'Sunki Maravilha' (Citrus sunki) mandarin, which show contrasting tolerances to water deficits at the physiological and molecular levels. Changes in the abundance of 36 and 38 proteins in Rangpur lime and 'Sunki Maravilha' mandarin, respectively, were observed via LC/MS/MS in response to water deficit. Multivariate principal component analysis (PCA) of the data revealed major changes in the protein profile of 'Sunki Maravilha' in response to water deficit. Additionally, proteomics and systems biology analyses allowed for the general elucidation of the major mechanisms associated with the differential responses to water deficit of both varieties. The defense mechanisms of Rangpur lime included changes in the metabolism of carbohydrates and amino acids as well as in the activation of reactive oxygen species (ROS) detoxification and in the levels of proteins involved in water stress defense. In contrast, the adaptation of 'Sunki Maravilha' to stress was aided by the activation of DNA repair and processing proteins. Our study reveals that the levels of a number of proteins involved in various cellular pathways are affected during water deficit in the roots of citrus plants. The results show that acclimatization to water deficit involves specific responses in Rangpur lime and 'Sunki Maravilha' mandarin. This study provides insights into the effects of drought on the abundance of proteins in the roots of two varieties of citrus rootstocks. In addition, this work allows for a better understanding of the

Protein-enhanced soups: a consumer-accepted food for increasing dietary protein provision among older adults.

Science.gov (United States)

Donahue, Elizabeth; Crowe, Kristi Michele; Lawrence, Jeannine

2015-02-01

Protein-enhanced soups (PES) may improve protein intake among older adults. This study examined sensory attributes (aroma, texture, taste, and overall acceptability) and preferences of PES (chicken noodle and cheddar broccoli) compared with flavor-matched control soups (FCS) among older adults (≥65 years) and evaluated dietary profile changes of a standard menu based on the substitution of one PES serving/d for a standard soup. Modified paired preference tests and 5-point facial hedonic scales were administered to participants (n = 44). No significant differences in sensory attributes between either PES compared with FCS were identified, but significant gender- and age-related differences (p preferred protein-enhanced chicken noodle soup while only 38% preferred protein-enhanced cheddar broccoli soup to their respective FCS. Substituting one PES serving for one non-fortified soup serving per day resulted in significantly higher (p < 0.001) protein profile. Results suggest that all attributes of PES were consistent with sensory expectations and PES substitution could improve protein provision.

Acute myeloid leukaemia: expression of MYC protein and its association with cytogenetic risk profile and overall survival.

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Mughal, Muhammad Kashif; Akhter, Ariz; Street, Lesley; Pournazari, Payam; Shabani-Rad, Meer-Taher; Mansoor, Adnan

2017-09-01

Acute myeloid leukaemia (AML) is a clinically aggressive disease with marked genetic heterogeneity. Cytogenetic abnormalities provide the basis for risk stratification into clinically favourable, intermediate, and unfavourable groups. There are additional genetic mutations, which further influence the prognosis of patients with AML. Most of these result in molecular aberrations whose downstream target is MYC. It is therefore logical to study the relationship between MYC protein expression and cytogenetic risk groups. We studied MYC expression by immunohistochemistry in a large cohort (n = 199) of AML patients and correlated these results with cytogenetic risk profile and overall survival (OS). We illustrated differential expression of MYC protein across various cytogenetic risk groups (p = 0.03). Highest expression of MYC was noted in AML patients with favourable cytogenetic risk group. In univariate analysis, MYC expression showed significant negative influence of OS in favourable and intermediate cytogenetic risk group (p = 0.001). Interestingly, MYC expression had a protective effect in the unfavourable cytogenetic risk group. In multivariate analysis, while age and cytogenetic risk group were significant factors influencing survival, MYC expression by immunohistochemistry methods also showed some marginal impact (p = 0.069). In conclusion, we have identified differential expression of MYC protein in relation to cytogenetic risk groups in AML patients and documented its possible impact on OS in favourable and intermediate cytogenetic risk groups. These preliminary observations mandate additional studies to further investigate the routine clinical use of MYC protein expression in AML risk stratification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Serum protein profile at remission can accurately assess therapeutic outcomes and survival for serous ovarian cancer.

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Jinhua Wang

Full Text Available BACKGROUND: Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer. EXPERIMENTAL DESIGN: Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD, remission (RM and recurrence (RC. The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis. RESULTS: Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC have individually good area-under-the-curve (AUC values (AUC = 0.69-0.86 and more than 10 three-marker combinations have excellent AUC values (0.91-0.93 in distinguishing active cancer samples (PD & RC from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC. Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1 measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10(-3. CONCLUSION: We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.

Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

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Jing Li

Full Text Available Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects.

Alterations in membrane protein-profile during cold treatment of alfalfa

International Nuclear Information System (INIS)

Mohapatra, S.S.; Poole, R.J.; Dhindsa, R.S.

1988-01-01

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [ 35 S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo

Modulating protein release profiles by incorporating hyaluronic acid into PLGA microparticles Via a spray dryer equipped with a 3-fluid nozzle

DEFF Research Database (Denmark)

Wan, Feng; Maltesen, Morten Jonas; Andersen, Sune Klint

2014-01-01

with or without HA were prepared using a spray dryer equipped with a 3-fluid nozzle. The effects of HA on the surface tension and the rheological behavior of the inner feed solution were investigated. The physicochemical properties of the resulting microparticles were characterized using scanning electron......: The present work demonstrates the potential of HA to modulate protein release profile from PLGA microparticle formulations produced via spray drying using 3-fluid nozzle....

Limiting factors in Escherichia colifed-batch production of recombinant proteins

DEFF Research Database (Denmark)

Sanden, A.M.; Prytz, I.; Tubelekas, I.

2003-01-01

recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation......recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation...

Modified expression of several sperm proteins after chronic exposure to the antiandrogenic compound vinclozolin.

Science.gov (United States)

Auger, Jacques; Eustache, Florence; Maceiras, Paula; Broussard, Cédric; Chafey, Philippe; Lesaffre, Corinne; Vaiman, Daniel; Camoin, Luc; Auer, Jana

2010-10-01

Little is known about the molecular impact of in vivo exposure to endocrine disruptors (EDs) on sperm structures and functions. We recently reported that the lifelong exposure of rats to the antiandrogenic compound vinclozolin results in low epididymal weight, changes in sperm kinematic parameters, and immature sperm chromatin condensation, together with the impairment of several fertility end points. These results led us to focus specifically on possible molecular abnormalities in sperm. Sperm samples were recovered from the frozen epididymides of rats exposed during the previous study. The proteins present in the samples from six exposed and six control rats were analyzed in pairs, by two-dimensional fluorescence difference gel electrophoresis, to investigate possible exposure-induced changes to sperm protein profiles. Twelve proteins, from the 380 matched spots observed in at least five gels, were present in larger or smaller amounts after vinclozolin exposure. These proteins were identified by mass spectrometry, and several are known to play a crucial role in the sperm fertilizing ability, among which, two mitochondrial enzymes, malate dehydrogenase 2 and aldehyde dehydrogenase (both of which were present in smaller amounts after treatment) and A-kinase anchor protein 4 (larger amounts of precursor after treatment). Finally, Ingenuity Pathway Analysis revealed highly significant interactions between proteins over- and underexpressed after treatment. This is the first study to show an association between in vivo exposure to an ED and changes to the sperm protein profile. These modifications may be at least partly responsible for the reproductive abnormalities and impaired fertility recently reported in this rat model of vinclozolin exposure.

Symmetric lens with extended depth of focus

OpenAIRE

Cho, Sung Nae

2008-01-01

The lens surface profile is derived based on the instantaneous focal length versus the lens radius data. The lens design based on instantaneous focal length versus the lens radius data has many useful applications in software assisted image focusing technology.

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

Science.gov (United States)

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

2009-09-01

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

Metabolic profile in different cathegories of diary cowst

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Ulchar Igor

2008-11-01

Full Text Available Knowing the values of the metabolic profile is important for prevention of so-called "production diseases" which have significant negative impact on the milk production. The aim of these investigations was determination of the metabolic profile and its referent values in the diary cows in several farms. Investigations included four categories of diary cows: pregnant heifers, cows in early lactation, cows in late lactation and dry cows. Discussion explains both significant differences of values of some parameters (glucosis, total proteins, urea, creatinine, AST, GGT in different groups of animals which were investigated and significant correlations between some parameters (glucosis, total protein, albumin, calcium, phosphorus, urea, creatinine, cholesterol, triglycerids, AST, GGT within each of groups of animals. The values gained with our investigations were compared with the referent values. It was found that cows included in our investigations were good metabolic profile, although their values were in some degree different from the referent values. Cows which were in lactation, especially the early lactation, had disposition of development of energy-protein deficit, but the disposition to calcium deficit was low.

Transcriptional profiling of putative human epithelial stem cells

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Koçer Salih S

2008-07-01

Full Text Available Abstract Background Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells. Results Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells. Conclusion This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells

Effect of Ramadan fasting on serum heat shock protein 70 and serum lipid profile.

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Zare, A; Hajhashemi, M; Hassan, Z M; Zarrin, S; Pourpak, Z; Moin, M; Salarilak, S; Masudi, S; Shahabi, S

2011-07-01

Ramadan, the holy month for the Islamic world, is a period every year when food and fluid intake is restricted to the pre-sunrise and post-sunset hours. The aim of this study was to evaluate the effect of Ramadan fasting on the serum concentration of heat shock protein 70 (HSP70) and serum lipid profile in healthy men. A total of 32 male volunteers with a mean age of 28.5 (range 23-37) years were selected for the study. Blood samples were obtained one day prior to Ramadan and on the 3rd and 25th days of fasting. Serum HSP70, triglyceride (TG), cholesterol (Chol), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), LDL/HDL and Chol/HDL ratios were investigated. It was observed that the mean concentrations of serum HSP70 and HDL on the 25th day of Ramadan were significantly higher than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels on the 3rd day of Ramadan was significantly higher than those recorded one day before Ramadan. Mean concentrations of serum TG, Chol, LDL, and LDL/HDL and Chol/HDL ratios on the 25th day of Ramadan were significantly lower than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels found on the 3rd day of Ramadan were also significantly lower than those recorded one day before Ramadan. Ramadan fasting increases serum HSP70 and improves serum lipid profile.

Effect of Doublesynch and Estradoublesynch protocols on estrus induction, conception rate, plasma progesterone, protein, and cholesterol profile in anestrus Gir heifers

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N. J. Chaudhary

2018-04-01

Full Text Available Aim: This study aimed to evaluate the efficacy of Doublesynch and Estradoublesynch protocols on estrus induction, conception rates, plasma progesterone, protein, and cholesterol profile in anestrus Gir heifers. Materials and Methods: In this study, 50 pubertal anestrus Gir heifers were selected from the field and farm conditions. The heifers were dewormed (injection ivermectin, 100 mg, s/c and supplemented with minerals and vitamins (injection organic phosphorus 800 mg and injection Vitamin AD3E and Biotin 10 ml i/m and multi-mineral bolus at 1 bolus daily for 7 days. The heifers were randomly divided into three groups: Doublesynch (n=20, Estradoublesynch (n=20, and control (n=10. The animals were monitored for estrus response, estrus interval, behavioral signs, and conception rates after induced/first, second, and third cycle post-treatment. Blood samples were obtained on day 0, day 9, day 12, and on day 12 post-artificial insemination (AI for determination of plasma progesterone, protein, and cholesterol profile. Results: The estrus response rate between Doublesynch and Estradoublesynch protocols was similar between treated heifers (85% and 95%. The interval from the second prostaglandin F2α (PGF2α injection to estrus induction did not differ between the groups (63.87±4.19 vs. 58.27±3.83 h. The conception rates following induced estrus (20% vs. 30%, at the second cycle (23.07% vs. 16.66%, at the third cycle (22.22% vs. 30.00%, and the overall conception rate (45% and 55% within 27.89±5.75 and 26.45±5.48 days were the same across the treatment groups. The mean plasma progesterone concentrations were significantly (p<0.01 higher on day 9 (second PGF2α injection and day 12 post-AI compared to day 0 (first PGF2α injection and the day of fixed-timed artificial insemination. The concentrations were also significantly (p<0.05 higher in conceived than non-conceived heifers on day 9 of treatment and day 12 post-AI in both the protocols. The

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

Energy Technology Data Exchange (ETDEWEB)

Zhen, Zhen [Shenzhen Institute, Peking University, Shenzhen 518057 (China); College of Engineering, Peking University, Beijing 100871 (China); Luthringer, Bérengère, E-mail: berengere.luthringer@hzg.de [Institute of Material Research, Helmholtz-Zentrum Geesthacht, Hamburg 21502 (Germany); Yang, Li [College of Engineering, Peking University, Beijing 100871 (China); Xi, Tingfei, E-mail: xitingfei@pku.edu.cn [Shenzhen Institute, Peking University, Shenzhen 518057 (China); Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Zheng, Yufeng [Shenzhen Institute, Peking University, Shenzhen 518057 (China); College of Engineering, Peking University, Beijing 100871 (China); Feyerabend, Frank; Willumeit, Regine [Institute of Material Research, Helmholtz-Zentrum Geesthacht, Hamburg 21502 (Germany); Lai, Chen [Shenzhen Institute, Peking University, Shenzhen 518057 (China); Ge, Zigang [College of Engineering, Peking University, Beijing 100871 (China)

2016-12-01

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48 h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48 h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. - Highlights: • First proteomic analysis of bioeffect mechanism caused by biodegradable Mg • Totally 867 proteins were identified by iTRAQ LC-MS/MS in this work. • 65 proteins were focused on because they were regulated within all the culture time. • The 65 proteins were associated with diverse biological processes. • Cell proliferation mechanism in Mg extract was investigated.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

International Nuclear Information System (INIS)

Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

2016-01-01

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48 h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48 h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. - Highlights: • First proteomic analysis of bioeffect mechanism caused by biodegradable Mg • Totally 867 proteins were identified by iTRAQ LC-MS/MS in this work. • 65 proteins were focused on because they were regulated within all the culture time. • The 65 proteins were associated with diverse biological processes. • Cell proliferation mechanism in Mg extract was investigated.

Influence of supplemental vitamin C on postmortem protein degradation and fatty acid profiles of the longissimus thoracis of steers fed varying concentrations of dietary sulfur.

Science.gov (United States)

Pogge, Danielle J; Lonergan, Steven M; Hansen, Stephanie L

2014-02-01

The objective was to examine the effects of supplemental vitamin C (VC) on postmortem protein degradation and fatty acid profiles of cattle receiving varying concentrations of dietary sulfur (S). A longissimus muscle was collected from 120 Angus-cross steers assigned to a 3 × 2 factorial, evaluating three concentrations of dietary S (0.22, 0.34, and 0.55%) and two concentrations of supplemental VC (0 or 10 g h(-1)d(-1)). Increasing dietary S and VC supplementation (Pdegradation (P = 0.07) and protein carbonylation (Pdegradation. © 2013.

Seldi-tof MS Profiling of Plasma Proteins in Ovarian Cancer

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Shao-Pai Wu

2006-03-01

Conclusion: This study clearly demonstrates that the combined technology of SELDI-TOF MS and artificial intelligence is effective in distinguishing protein expression between normal and ovarian cancer plasma. The identified protein peaks may be candidate proteins for early detection of ovarian cancer or evaluation of therapeutic response.

Optimization of translation profiles enhances protein expression and solubility.

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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.

Optimal Focusing and Scaling Law for Uniform Photo-Polymerization in a Thick Medium Using a Focused UV Laser

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Jui-Teng Lin

2014-02-01

Full Text Available We present a modeling study of photoinitiated polymerization in a thick polymer-absorbing medium using a focused UV laser. Transient profiles of the initiator concentration at various focusing conditions are analyzed to define the polymerization boundary. Furthermore, we demonstrate the optimal focusing conditions that yield more uniform polymerization over a larger volume than the collimated or non-optimal cases. Too much focusing with the focal length f < f* (an optimal focal length yields a fast process; however, it provides a smaller polymerization volume at a given time than in the optimal focusing case. Finally, a scaling law is derived and shows that f* is inverse proportional to the product of the extinction coefficient and the initiator initial concentration. The scaling law provides useful guidance for the prediction of optimal conditions for photoinitiated polymerization under a focused UV laser irradiation. The focusing technique also provides a novel and unique means for obtaining uniform photo-polymerization within a limited irradiation time.