Exosome-associated release, uptake, and neurotoxicity of HIV-1 Tat protein.

PubMed

Rahimian, Pejman; He, Johnny J

2016-12-01

HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.

Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

PubMed Central

Palmer, Ira; Wingfield, Paul T.

2013-01-01

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are not restricted to E. coli; they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up ~60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine·HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT 6.5 or further purified by gel filtration in the presence of guanidine·HCl as in basic Protocol 2. A Support Protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE (UNIT 10.1). Other methods discussed in the Commentary section of this unit include the direct purification of polyhistidine-tagged proteins solubilized in guanidine·HCl, preparative removal of guanidine·HCl by reversed-phase chromatography as a prelude to protein folding, and the solubilization of inclusion bodies with anionic detergents. PMID:23151747

Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

PubMed Central

Palmer, Ira; Wingfield, Paul T.

2012-01-01

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; however, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates by low speed centrifugation. Following preextaction (or washing) protein is extracted from washed pellets using guanidine·HCl. The solubilized and unfolded protein is either directly folded as described in UNIT 6.1 or further purified by gel filtration in the presence of guanidine·HCl as described here. A support protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE. High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies (UNITS 5.1 & 6.1). Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies are not restricted to E. coli; they can also form in yeast, mammalian, and insect cells. Inclusion bodies recovered from cell lysates by low-speed centrifugation are heavily contaminated with E. coli cell wall and outer membrane components. The latter are largely removed by selective extraction with detergents and low concentrations of either urea or guanidine·HCl to produce so-called washed pellets. These basic steps result in a significant purification of the recombinant protein, which usually makes up ~60% of the washed pellet protein. The challenge, therefore, is not to purify the recombinant-derived protein, but to solubilize it and then fold it into native and biologically active protein. Basic Protocol 1 describes preparation of washed pellets and solubilization of the protein using guanidine·HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT 6.5 or further purified by gel filtration in the presence of guanidine·HCl as in basic Protocol 2. A Support Protocol describes the removal of guanidine·HCl from column fractions so they can be monitored by SDS-PAGE (UNIT 10.1). Other methods discussed in the Commentary section of this unit include the direct purification of polyhistidine-tagged proteins solubilized in guanidine·HCl, preparative removal of guanidine·HCl by reversed-phase chromatography as a prelude to protein folding, and the solubilization of inclusion bodies with anionic detergents. PMID:18429271

Changes in rat parotid saliva protein composition following chronic reserpine treatment and their relation to inanition.

PubMed

Johnson, D A

1988-01-01

Chronic administration of the catecholamine-depleting agent, reserpine (0.5 mg/kg), resulted in a reduction in food intake after 3 days. To differentiate effects of the drug from those of reduced food intake a pair-fed group, whose daily caloric intake was restricted to the amount consumed by the reserpine-treated rats, was included. After 7 days, both the reserpine-treated and pair-fed control exhibited a marked reduction in the volume of saliva collected in a 30 min interval following a secretory stimulus compared to untreated ad libitum-fed controls, and the proportion of salivary proteins attributable to acidic and basic proline-rich proteins and to minor 1b protein were decreased whereas deoxyribonuclease was increased. For two of the salivary proteins (fractions I and V) changes for the reserpine-treated and pair-fed groups were different. Fraction I was reduced in both groups, but exhibited a greater decrease in the pair-fed than in the reserpine-treated, whereas fraction V was significantly increased only in the pair-fed group. Thus many of the salivary changes associated with reserpine treatment may have resulted from the change in feeding habits and not from reserpine treatment per se. The study demonstrates the importance of controlling for food intake under experimental circumstances which may lead to a marked change in daily feeding habits.

Fractional vector calculus for fractional advection dispersion

NASA Astrophysics Data System (ADS)

Meerschaert, Mark M.; Mortensen, Jeff; Wheatcraft, Stephen W.

2006-07-01

We develop the basic tools of fractional vector calculus including a fractional derivative version of the gradient, divergence, and curl, and a fractional divergence theorem and Stokes theorem. These basic tools are then applied to provide a physical explanation for the fractional advection-dispersion equation for flow in heterogeneous porous media.

Screening and comparison of antioxidant activities of polysaccharides from Coriolus versicolor.

PubMed

Sun, Xiaowen; Sun, Yanping; Zhang, Qingbo; Zhang, Hongwei; Yang, Bingyou; Wang, Zhibin; Zhu, Weiguo; Li, Bin; Wang, Qiuhong; Kuang, Haixue

2014-08-01

Six polysaccharide fractions (Coriolus versicolor polysaccharides: CVPS-1, CVPS-2, CVPS-3, CVPS-4, CVPS-5 and CVPS-6) were isolated and purified from the fruiting bodies of C. versicolor by ion exchange chromatography and gel chromatography. Their chemical and physical characteristics were determined by chemical methods, high performance liquid chromatography, and high-performance gel-permeation chromatography. Finally, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, superoxide radical assay, and hydroxyl radical assay were carried out to test the antioxidant activities of CVPS in vitro. The results indicated that the six CVPS fractions were acidic heteropolysaccharides, composed of mannose, rhamnose, glucuronic acid, glucose and fructose with different ratios. The molecular weights of CVPS-1, CVPS-2, CVPS-3, CVPS-4, CVPS-5 and CVPS-6 were 1740, 1480, 568, 880, 1260 and 1840kDa and the protein contents were 4.2%, 6.4%, 8.5%, 7.8%, 6.5% and 3.9%, respectively. Among the six fractions, CVPS with lower molecular weight, higher protein content and larger uronic acid amount, basically exhibited higher radical scavenging effects at the same concentration. Compared with other fractions, CVPS-3 exhibited the highest antioxidant activities. The effects of the molecular weight, protein content and uronic acid amount of the polysaccharides appeared to be significant on the improvement of the bioactivities. Copyright © 2014 Elsevier B.V. All rights reserved.

A Proteomic Analysis of the Body Wall, Digestive Tract, and Reproductive Tract of Brugia malayi

PubMed Central

Morris, C. Paul; Bennuru, Sasisekhar; Kropp, Laura E.; Zweben, Jesse A.; Meng, Zhaojing; Taylor, Rebekah T.; Chan, King; Veenstra, Timothy D.; Nutman, Thomas B.; Mitre, Edward

2015-01-01

Filarial worms are parasitic nematodes that cause devastating diseases such as lymphatic filariasis (LF) and onchocerciasis. Filariae are nematodes with complex anatomy including fully developed digestive tracts and reproductive organs. To better understand the basic biology of filarial parasites and to provide insights into drug targets and vaccine design, we conducted a proteomic analysis of different anatomic fractions of Brugia malayi, a causative agent of LF. Approximately 500 adult female B. malayi worms were dissected, and three anatomical fractions (body wall, digestive tract, and reproductive tract) were obtained. Proteins from each anatomical fraction were extracted, desalted, trypsinized, and analyzed by microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry. In total, we identified 4,785 B. malayi proteins. While 1,894 were identified in all three anatomic fractions, 396 were positively identified only within the digestive tract, 114 only within the body wall, and 1,011 only within the reproductive tract. Gene set enrichment analysis revealed a bias for transporters to be present within the digestive tract, suggesting that the intestine of adult filariae is functional and important for nutrient uptake or waste removal. As expected, the body wall exhibited increased frequencies of cytoskeletal proteins, and the reproductive tract had increased frequencies of proteins involved in nuclear regulation and transcription. In assessing for possible vaccine candidates, we focused on proteins sequestered within the digestive tract, as these could possibly represent “hidden antigens” with low risk of prior allergic sensitization. We identified 106 proteins that are enriched in the digestive tract and are predicted to localize to the surface of cells in the the digestive tract. It is possible that some of these proteins are on the luminal surface and may be accessible by antibodies ingested by the worm. A subset of 27 of these proteins appear especially promising vaccine candidates as they contain significant non-cytoplasmic domains, only 1–2 transmembrane domains, and a high degree of homology to W. bancrofti and/or O. volvulus. PMID:26367142

Novel peptides from adrenomedullary chromaffin vesicles.

PubMed Central

Sigafoos, J; Chestnut, W G; Merrill, B M; Taylor, L C; Diliberto, E J; Viveros, O H

1993-01-01

The adrenal medulla chromaffin vesicle (CV) contains, on a weight basis, as much soluble protein and peptide as catecholamine. The bulk of the protein is accounted for by chromogranins (Cgr) A, B and C. Additionally, a large variety of neuropeptides and their precursor proteins have been found recently within these vesicles. Nevertheless, fractionation of CV lysates indicates the presence of many more peptides than previously reported. In the hope of finding novel bioactive peptides, we initiated a systematic isolation and characterisation of CV peptides. Bovine CV pellets were prepared by sucrose gradient centrifugation and immediately boiled in water to avoid degradation of native proteins and peptides. The water lysates were fractionated through a battery of reversed-phase and ion-exchange high-performance chromatographic steps. We fully or partially characterised a substantial number of novel peptides derived from CgrA and CgrB. A tetradecapeptide and a 13 kDa extended peptide were derived from the bovine homologue of rat secretogranin III. Peptides corresponding to C-terminal fragments of 7B2 and proteoglycan II were also found. Additionally, several sequences had no known precursors. Of the sequences derived from known precursors some corresponded to fragments bracketed by pairs of basic amino acids, but others were preceded or followed by single basic residues or by unusual putative cleavage sites. Some of these peptides were postranslationally modified (pyroglutamylation, glycosylation, phosphorylation, amidation). A significant degree of structural conservation of some of these peptides across species suggests that they may exert biological effects when cosecreted with catecholamines during splanchnic stimulation. PMID:8300415

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung

Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau proteinmore » structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.« less

Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

PubMed Central

Enyaru, John C.; Carr, Steven A.; Pearson, Terry W.

2013-01-01

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification. PMID:23951171

Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

PubMed

Eyford, Brett A; Ahmad, Rushdy; Enyaru, John C; Carr, Steven A; Pearson, Terry W

2013-01-01

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a 'deep-mining" proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.

Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development.

PubMed

Lesage, Véronique S; Merlino, Marielle; Chambon, Christophe; Bouchet, Brigitte; Marion, Didier; Branlard, Gérard

2012-01-01

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics.

PubMed

Li, Zhao Bo; Flint, Paul W; Boluyt, Marvin O

2005-09-01

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.

Biodegradation of composite resin with ester linkages: identifying human salivary enzyme activity with a potential role in the esterolytic process.

PubMed

Cai, Kuihua; Delaviz, Yasaman; Banh, Michael; Guo, Yi; Santerre, J Paul

2014-08-01

The ester linkages contained within dental resin monomers (such as Bisphenol A-glycidylmethacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA)) are susceptible to hydrolytic degradation by salivary esterases, however very little is known about the specific esterase activities implicated in this process. The objective of this work was to isolate and identify the dominant proteins from saliva that are associated with the esterase activities shown to be involved in the degradation of BisGMA. Human whole saliva was collected and processed prior to separation in a HiPrep 16/60 Sephacryl S-200 HR column. The fraction with the highest esterase activity was further separated by an anion exchange column (Mono-Q (10/100G)). Isolated fractions were then separated by gel electrophoresis, and compared to a common bench marker esterase, cholesterol esterase (CE), and commercial albumin which has been reported to express esterase activity. Proteins suspected of containing esterase activity were analyzed by Mass Spectroscopy (MS). Commercially available proteins, similar to the salivary esterase proteins identified by MS, were used to replicate the enzymatic complexes and confirm their degradation activity with respect to BisGMA. MS data suggested that the enzyme fraction with the highest esterase activity was contained among a group of proteins consisting of albumin, Zn-α2-glycoprotein, α-amylase, TALDO1 protein, transferrin, lipocalin2, and prolactin-induced protein. Studies concluded that the main esterase bands on the gels in each fraction did not overlap with CE activity, and that albumin activity emerged as a lead candidate with significant esterase activity relative to BisGMA degradation, particularly when it formed a complex with Zn-α2-glycoprotein, under slightly basic conditions. These enzyme complexes can be used as a physiologically relevant formulation to test the biostability of composite resins. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Fractional Poisson-Nernst-Planck Model for Ion Channels I: Basic Formulations and Algorithms.

PubMed

Chen, Duan

2017-11-01

In this work, we propose a fractional Poisson-Nernst-Planck model to describe ion permeation in gated ion channels. Due to the intrinsic conformational changes, crowdedness in narrow channel pores, binding and trapping introduced by functioning units of channel proteins, ionic transport in the channel exhibits a power-law-like anomalous diffusion dynamics. We start from continuous-time random walk model for a single ion and use a long-tailed density distribution function for the particle jump waiting time, to derive the fractional Fokker-Planck equation. Then, it is generalized to the macroscopic fractional Poisson-Nernst-Planck model for ionic concentrations. Necessary computational algorithms are designed to implement numerical simulations for the proposed model, and the dynamics of gating current is investigated. Numerical simulations show that the fractional PNP model provides a more qualitatively reasonable match to the profile of gating currents from experimental observations. Meanwhile, the proposed model motivates new challenges in terms of mathematical modeling and computations.

Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers.

PubMed

Heebøll-Nielsen, Anders; Justesen, Sune F L; Thomas, Owen R T

2004-09-30

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.042 microM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-exchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl in the elution buffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (< or = 0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was increased to 0.4-1 M. The anion-exchanger adsorbed beta-lactoglobulin (beta-LG) selectively allowing separation from the remaining protein.

Phosphoinositide-binding proteins in autophagy.

PubMed

Lystad, Alf Håkon; Simonsen, Anne

2016-08-01

Phosphoinositides represent a very small fraction of membrane phospholipids, having fast turnover rates and unique subcellular distributions, which make them perfect for initiating local temporal effects. Seven different phosphoinositide species are generated through reversible phosphorylation of the inositol ring of phosphatidylinositol (PtdIns). The negative charge generated by the phosphates provides specificity for interaction with various protein domains that commonly contain a cluster of basic residues. Examples of domains that bind phosphoinositides include PH domains, WD40 repeats, PX domains, and FYVE domains. Such domains often display specificity toward a certain species or subset of phosphoinositides. Here we will review the current literature of different phosphoinositide-binding proteins involved in autophagy. © 2016 Federation of European Biochemical Societies.

Ion-exchange chromatography: basic principles and application to the partial purification of soluble mammalian prolyl oligopeptidase.

PubMed

Cummins, Philip M; Dowling, Oonagh; O'Connor, Brendan F

2011-01-01

Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

Sources of Group and Individual Differences in Emerging Fraction Skills

PubMed Central

Hecht, Steven A.; Vagi, Kevin J.

2010-01-01

Results from a two year longitudinal study of 181 children from fourth through fifth grade are reported. Levels of growth in children’s computation, word problem, and estimation skills using common fractions were predicted by working memory, attentive classroom behavior, conceptual knowledge about fractions, and simple arithmetic fluency. Comparisons of 55 participants identified as having mathematical difficulties to those without mathematical difficulties revealed that group differences in emerging fraction skills were consistently mediated by attentive classroom behavior and conceptual knowledge about fractions. Neither working memory nor arithmetic fluency mediated group differences in growth in fraction skills. It was also found that the development of basic fraction skills and conceptual knowledge are bidirectional in that conceptual knowledge exerted strong influences on all three types of basic fraction skills, and basic fraction skills exerted a more modest influence on subsequent conceptual knowledge. Results are discussed with reference to how the identification of potentially malleable student characteristics that contribute to the difficulties that some students have with fractions informs interventions and also will contribute to a future theoretical account concerning how domain general and domain specific factors influence the development of basic fraction skills. PMID:21170171

7 CFR 1405.2 - Basic rule of fractions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Basic rule of fractions. 1405.2 Section 1405.2 Agriculture Regulations of the Department of Agriculture (Continued) COMMODITY CREDIT CORPORATION, DEPARTMENT... rule of fractions. Fractions shall be rounded in accordance with the provisions of 7 CFR part 718. ...

Studies on sex-organ development. Changes in nuclear and chromatin composition and genomic activity during spermatogenesis in the maturing rooster testis.

PubMed Central

Mezquita, C; Teng, C S

1977-01-01

We developed a technique to separate nuclei of rooster testis by centrifugation through a discontinuous sucrose density gradient and by sedimentation at unit gravity. Four different major fractions obtained from testicular nuclei and one from the vas deferens were characterized according to their velocity of sedimentation, morphology and DNA content. The ratios (w/w) of basic proteins, non-histone proteins and RNA to DNA decreased during spermiogenesis both in nuclei and chromatin. Changes in the electrophoretic patterns of histones and non-histone proteins were detected especially in the elongated spermatids. The lack of uptake of [3H]uridine in elongating and elongated spermatids and in spermatozoa was demonstrated by radioautography and by the detection of labelled RNA extracted from different fractions of nuclei. Template activity for RNA synthesis and the binding of actinomycin D by testicular nuclei reached a peak in the elongated spermatid stage, when the histones are replaced by the protamine. Images PLATE 1 PLATE 2 PLATE 3 PLATE 4 PLATE 5 PMID:560187

Basic Theory of Fractional Conformal Invariance of Mei Symmetry and its Applications to Physics

NASA Astrophysics Data System (ADS)

Luo, Shao-Kai; Dai, Yun; Yang, Ming-Jing; Zhang, Xiao-Tian

2018-04-01

In this paper, we present a basic theory of fractional dynamics, i.e., the fractional conformal invariance of Mei symmetry, and find a new kind of conserved quantity led by fractional conformal invariance. For a dynamical system that can be transformed into fractional generalized Hamiltonian representation, we introduce a more general kind of single-parameter fractional infinitesimal transformation of Lie group, the definition and determining equation of fractional conformal invariance are given. And then, we reveal the fractional conformal invariance of Mei symmetry, and the necessary and sufficient condition whether the fractional conformal invariance would be the fractional Mei symmetry is found. In particular, we present the basic theory of fractional conformal invariance of Mei symmetry and it is found that, using the new approach, we can find a new kind of conserved quantity; as a special case, we find that an autonomous fractional generalized Hamiltonian system possesses more conserved quantities. Also, as the new method's applications, we, respectively, find the conserved quantities of a fractional general relativistic Buchduhl model and a fractional Duffing oscillator led by fractional conformal invariance of Mei symmetry.

Binding of DNA-bending non-histone proteins destabilizes regular 30-nm chromatin structure

PubMed Central

Bajpai, Gaurav; Jain, Ishutesh; Inamdar, Mandar M.; Das, Dibyendu; Padinhateeri, Ranjith

2017-01-01

Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure—a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions. PMID:28135276

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties.

PubMed

Mo, Xiaoqun; Zhong, Zhikai; Wang, Donghai; Sun, Xiuzhi

2006-10-04

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins.

PubMed

del Valle, Luis J

2005-06-01

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.

Protein methylation in pea chloroplasts. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niemi, K.J.; Adler, J.; Selman, B.R.

1990-07-01

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with ({sup 3}H-methyl)-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. Onemore » methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile ({sup 3}H)methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the ({sup 3}H)methyl group.« less

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

PubMed

Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

2005-07-01

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia.

PubMed

Kapprell, H P; Owaribe, K; Franke, W W

1988-05-01

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.

Cottonseed protein derivatives as nutritional and functional supplements in food formulations.

PubMed

Cherry, J P; Berardi, L C; Zarins, Z M; Wadsworth, J I; Vinnett, C H

1978-01-01

Cottonseeds contain protein with desirable food functional and nutritional properties. Storage globulins make up most of the protein stored in cottonseed and can be separated into five fractions by gel filtration chromatography. Each fraction is distinguishable from the other by its amino acid and polyacrylamide gel electrophoretic properties. Proteins of cottonseed contribute greatly to the functional properties of emulsions, co-isolates, and texturized derivatives. For example, increasing the amount of high protein cottonseed flour in wheat suspensions from 2% to 10% improved the capacity (54-97 ml of oil) and viscosity (5,000-100,000+ cps) of emulsions. The 10% suspension formed emulsions with increasing oil capacity (84-100 ml) and viscosity (28,000-100,000+ cps) as the pH was adjusted from 4.5 to 9.5. Consistencies of the products ranged from that of salad dressing (low percent suspensions, or acid pH) to that of mayonnaise (high percent, or basic pH). These data were utilized to derive a multiple regression model to predict optimum use of cottonseed proteins in emulsions of varying consistencies. A coprecipitated isolate containing greater than 94% protein was prepared from a blend of cottonseed and peanut flours. Amino acid content of the co-isolate reflected that of the protein in the two flours of the composite. The co-isolate has lower gossypol level and improved color and functional properties than a cottonseed protein isolate. Storage protein isolate of cottonseed suspended in aqueous solution and heated with constant stirring forms a texturized product; the quality of the product depends on heat, pH, salt, and the quantity of nonstorage proteins. Protein and amino acid content of meat products were improved by the addition of the texturized protein of cottonseed.

Removal of natural organic matter from water using ion-exchange resins and cyclodextrin polyurethanes

NASA Astrophysics Data System (ADS)

Nkambule, T. I.; Krause, R. W.; Mamba, B. B.; Haarhoff, J.

Natural organic matter (NOM) consists of a complex mixture of naturally occurring organic compounds. Although it is not considered toxic by itself, NOM present during water disinfection may result in the formation of disinfection by-products (DBPs), many of which are either carcinogenic or mutagenic. Although it is difficult to completely characterize NOM due to its complex and large structure, a consideration of its structure is necessary for a better understanding of the mechanism of NOM removal from water. In this study, water from the Vaalkop water treatment plant was characterized for its NOM composition by fractionation over ion-exchange resins. Fractionation at different pH with different resins resulted in the isolation of the neutral, basic and acidic fractions of both the hydrophobic and hydrophilic NOM. The hydrophilic basic fraction was found to be the most abundant fraction in the source water. Each of the isolated NOM fractions were percolated through cyclodextrin (CD) polyurethanes, resulting in an adsorption efficiency of between 6% and 33%. The acidic fractions were the most adsorbed fractions by the CD polyurethanes, while the neutral fractions being the least adsorbed. The water samples were then subjected to an ozonation regime at the treatment plant and then fractionated as before. As expected there were decreases of the neutral and basic fractions after ozonation. The application of CD polyurethanes to the fractions after ozonation resulted in a removal efficiency of up to 59%, nearly double that of the non-treated sample. Also, in the case of the ozone pre-treated samples, it was mainly the hydrophilic basic fraction which was removed. All the fractions were subjected to a chlorination test to determine the trihalomethane (THM) formation potential. All six NOM fractions resulted in THM formation, but the hydrophilic basic fraction was found to be the most reactive and formed the highest THM concentration. The effect of the combination of ozone and cyclodextrin polyurethane resulted in a relatively good capability to remove NOM from water as evidenced by an up overall 88% reduction of the hydrophilic acidic fraction.

Fractional vector calculus and fluid mechanics

NASA Astrophysics Data System (ADS)

Lazopoulos, Konstantinos A.; Lazopoulos, Anastasios K.

2017-04-01

Basic fluid mechanics equations are studied and revised under the prism of fractional continuum mechanics (FCM), a very promising research field that satisfies both experimental and theoretical demands. The geometry of the fractional differential has been clarified corrected and the geometry of the fractional tangent spaces of a manifold has been studied in Lazopoulos and Lazopoulos (Lazopoulos KA, Lazopoulos AK. Progr. Fract. Differ. Appl. 2016, 2, 85-104), providing the bases of the missing fractional differential geometry. Therefore, a lot can be contributed to fractional hydrodynamics: the basic fractional fluid equations (Navier Stokes, Euler and Bernoulli) are derived and fractional Darcy's flow in porous media is studied.

Commercial breakfast cereals available in Mexican markets and their contribution in dietary fiber, β-glucans and protein quality by rat bioassays.

PubMed

Falcón-Villa, María R; Barrón-Hoyos, Jesús M; Cinco-Moroyoqui, Francisco J

2014-09-01

The beneficial effect of dietary fiber (DF) consumption has long been recognized. The global economy and open market trade policies have increased the availability of food products in Mexican markets, resulting in a wide variety of ready-to-eat commercial breakfast cereals classified as 'high fiber'. This research was aimed to evaluate the total dietary fiber contents, its fractions (soluble and insoluble) and β-glucan in 13 commercial 'high-fiber' breakfast cereals, as well as to evaluate their protein quality by rat bioassays. Commercial 'high-fiber' breakfast cereals had 7.42-39.82% insoluble dietary fiber, 2.53-12.85% soluble dietary fiber, and 0.45-4.96% β-glucan. These ready-to-eat commercial 'high-fiber' breakfast cereals differed significantly in their total dietary fiber, their soluble and insoluble DF fractions, and also in their β-glucan contents. When supplied as experimental diets, in 14-day rat feeding trials, the 'high-fiber' breakfast cereals showed an adverse effect on the % N digestibility but protein utilization, as measured as net protein ratio (NPR), was not significantly affected. The consumption of these commercial breakfast cereals, especially those made of oats as the basic ingredient, is highly recommended, since these products, being a concentrated source of dietary fiber, do not affect their protein quality.

Mechanism of polyphosphate kinase from Propionibacterium shermanii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robinson, N.A.

1986-01-01

Polyphosphate kinase, which catalyzes the reaction shown below, is one of two enzymes which have been reported to catalyze the synthesis of polyphosphate. Purification performed by ammonium sulfate precipitation (0-40% fraction) was followed by chromatography. The enzyme represents 70% of the protein in the hydroxylapatite pool and is stable at this level of purity. The subunit molecular weight was determined by SDS polyacrylamide gel analysis, (83,000 +/- 3000), nondenaturing polyacrylamide gel electrophoresis, (80,000 and 86,000 daltons), gel filtration (Biogel A 0.5m column was 85,000 +/- 4000.) Polyphosphate kinase appears to be a monomeric enzyme of approx.83,000 daltons. Four assays weremore » developed for polyphosphate kinase. Basic proteins such as polylysine stimulate the synthesis of polyphosphate, these proteins cause precipitation of polyphosphate kinase from relatively impure enzyme extracts: Synthesized polyphosphate interacts noncovalently with the basic protein-enzyme precipitate. Efficient synthesis of polyphosphate requires the addition of either phosphate or short chain polyphosphate. Synthesis did occur at 1/10 the rate when neither of these two compounds were included. Initiation, elongation, and termination events of polyphosphate synthesis were examined. Short chain polyphosphate acts as a primer, with (/sup 32/P) short-chain polyphosphate incorporation into long chain polyphosphate by the kinase.« less

Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third

PubMed Central

Baxter, Michael K.; Gibson, Wade

2001-01-01

The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule—the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites. PMID:11435566

Whey protein solution coating for fat-uptake reduction in deep-fried chicken breast strips.

PubMed

Dragich, Ann M; Krochta, John M

2010-01-01

This study investigated the use of whey protein, as an additional coating, in combination with basic, well-described predust, batter, and breading ingredients, for fat-uptake reduction in fried chicken. Chicken breasts were cut into strips (1 x 5 x 10 cm) and coated with wheat flour (WF) as a predust, dipped in batter, coated with WF as a breading, then dipped in 10% denatured whey protein isolate (DWPI) aqueous solution (wet basis). A WF-batter-WF treatment with no DWPI solution dip was included as a control. Coated chicken strips were deep-fried at 160 degrees C for 5 min. A Soxhlet-type extraction was performed to determine the fat content of the meat fraction of fried samples, the coating fraction of fried samples, raw chicken, and raw coating ingredients. The WF-batter-WF-10% DWPI solution had significantly lower fat uptake than the WF-batter-WF control, by 30.67% (dry basis). This article describes applied research involving fat reduction in coated deep-fried chicken. The methods used in this article were intended to achieve maximized fat reduction while maintaining a simple procedure applicable to actual food processing lines.

The role of myelin lipids in experimental allergic encephalomyelitis. Part 1. Influence on disease production by non-encephalitogenic doses of myelin basic protein.

PubMed

Hosein, Z Z; Gilbert, J J; Strejan, G H

1984-12-01

Hartley guinea pig central nervous system (CNS) myelin has been purified and fractionated into its protein and lipid components. Experimental allergic encephalomyelitis (EAE) was induced in juvenile strain 13 guinea pigs with both lyophilized and fresh 'wet' myelin. However, a larger dose of lyophilized myelin was required to induce chronic EAE. Total myelin lipids, galactocerebrosides, gangliosides, phospholipids or proteolipids were combined with a non-encephalitogenic dose of myelin basic protein (MBP) and injected in juvenile Hartley guinea pigs. No clinical or histological manifestations of disease were observed. Parameters of immune functions indicated that the total myelin lipids augmented cell-mediated immune responses as measured by in vitro lymphocyte transformation and by a significant decrease in the percentage of peripheral early T cells. Only the proteolipids elicited delayed hypersensitivity reactions. Animals that received the phospholipid-MBP combination showed no changes when compared to animals injected with MBP alone. The results suggest that although the myelin lipids did not act synergistically with a non-encephalitogenic dose of MBP to induce EAE, they induced immunological changes and potentiated the immune response to MBP.

21 CFR 862.1630 - Protein (fractionation) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Protein (fractionation) test system. 862.1630... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation) test system is a device intended to measure protein fractions in blood, urine, cerebrospinal fluid, and other...

Chemical and functional properties of the different by-products of artichoke (Cynara scolymus L.) from industrial canning processing.

PubMed

Ruiz-Cano, Domingo; Pérez-Llamas, Francisca; Frutos, María José; Arnao, Marino B; Espinosa, Cristóbal; López-Jiménez, José Ángel; Castillo, Julián; Zamora, Salvador

2014-10-01

In this study, the basic chemical composition and functional properties of six by-product fractions collected from different steps of artichoke industrial processing were evaluated. Fractions differed in thermal treatment, the bract position in the artichoke head and the cutting size. Contents of moisture, ash, protein, fat, dietary fibre, inulin, total phenolics, total flavonoids, caffeoyl derivatives and flavones were analysed. Antioxidant activity values were also determined. All assessed artichoke by-product fractions contained high-dietary fibre (53.6-67.0%) and low fat (2.5-3.7%). Artichoke by-product fractions contained high levels of inulin, especially in the boiled inner bracts (30%). Total phenolic and flavonoid contents and antioxidant activity (153-729 μmol gallic acid equivalents, 6.9-19.2 μmol quercetin equivalents and 85-234 μmol ascorbic acid equivalents per gram of dry matter, respectively) varied widely with the bract positions in the artichoke head and the thermal treatments. The more interesting fractions for use as functional ingredients were those situated closer to the artichoke heart and thermally treated. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biochemical properties of a novel 28KDA protein tyrosine kinase partially purified from the particulate fraction of rat spleen.

PubMed

Borowski, P; Medem, S; Laufs, R

1993-12-15

In this report we present some of the biochemical properties of the enzyme, here called pp28(PTK), isolated from particulate fraction of rat spleen (1). The kinase is very susceptible for polyions as regulators of the enzymatic activity. The polyanions like dextran sulfate or heparin inhibited, and polycations such as spermidin, protamin, poly-L-lysine and some random polypeptides containing tyrosine besides a basic amino acid, stimulated the enzyme markedly. The kinase showed high sensitivity towards class IA salts. In the casein phosphorylation reaction the apparent Km value for ATP was 4 microM. An unusual property is associated with autophosphorylation which leads to a reduced activity towards external substrates. Some kinase inhibitors described in the literature were tested for their potency.

Toxicokinetics and toxicodynamics of chlorpyrifos is altered in embryos of Japanese medaka exposed to oil sands process-affected water: evidence for inhibition of P-glycoprotein.

PubMed

Alharbi, Hattan A; Alcorn, Jane; Al-Mousa, Ahmed; Giesy, John P; Wiseman, Steve B

2017-05-01

Oil sands process-affected water (OSPW) is generated during extraction of bitumen in the surface mining oil sands industry in Alberta, Canada. Studies were performed in vitro by use of Caco-2 cells, and in vivo with larvae of Japanese medaka (Oryzias latipes) to determine if organic compounds from the aqueous phase of OSPW inhibit ATP binding cassette protein ABCB1 (permeability-glycoprotein, P-gp). Neutral and basic fractions of OSPW inhibited activity of P-gp in Caco-2 cells by 1.9- and 2.0-fold, respectively, while the acidic fraction had the least effect. The organophosphate pesticides chlorpyrifos (a substrate of P-gp) and malathion (not a substrate of P-gp), were used as model chemicals to investigate inhibition of P-gp in larvae. Co-exposure to chlorpyrifos and an extract of OSPW containing basic and neutral compounds reduced survival of larvae to 26.5% compared to survival of larvae exposed only to chlorpyrifos, which was 93.7%. However, co-exposure to malathion and the extract of OSPW did not cause acute lethality compared to exposure only to malathion. Accumulation and bioconcentration of chlorpyrifos, but not malathion, was greater in larvae co-exposed with the extract of OSPW. The terminal elimination half-life of chlorpyrifos in larvae exposed to chlorpyrifos in freshwater was 5 days compared with 11.3 days in larvae exposed to chlorpyrifos in OSPW. Results suggest that in non-acute exposures, basic and neutral organic compounds in the water-soluble fraction of OSPW inhibit activity of P-gp, which suggests that OSPW has the potential to cause adverse effects by chemosensitization. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

PubMed Central

2010-01-01

Background Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry. Results Fifty-seven protein spots were excised from control group gels, and 21 different proteins were identified. With tannin consumption, the expression levels of one α-amylase isoform and one unidentified protein increased, whereas acidic mammalian chitinase and Muc10 decreased. Additionally, two basic spots that stained pink with Coomassie Brilliant Blue R-250 were newly observed, suggesting that some induced PRPs may remain uncomplexed or form soluble complexes with tannins. Conclusion This proteomic analysis provides evidence that other salivary proteins, in addition to tannin-precipitating proteins, are affected by tannin ingestion. Changes in the expression levels of the acidic mammalian chitinase precursor and in one of the 14 salivary α-amylase isoforms underscores the need to further investigate their role in tannin ingestion. PMID:21159160

Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species.

PubMed

Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Alodat, Moh'd; Al-Mahasneh, Majdi A; Gammoh, Sana; Ereifej, Khalil; Almajwal, Ali; Kubow, Stan

2017-09-01

This study aimed to investigate the molecular and bio-functional properties of protein fractions from Nigella damascena and Nigella arvensis, including the albumin, globulin, glutein-1, glutein-2 and prolamin fractions. Protein subunits were not observed in globulin and prolamin fractions. No peaks appeared in RP-HPLC chromatograms of globulin for either species. Two predominant peaks were observed in the RP-HPLC profiles of all protein fractions. Proteins separated by RP-HPLC have potential inhibitory and antioxidant activities in all fractions. Optimum ACE-inhibitory and antioxidant activities of proteins separated by RP-HPLC were observed in glutein-2 and albumin, respectively, for both species. For pepsin and combined pepsin-trypsin hydrolyses, the highest degree of hydrolysis (DH) was obtained in glutein-2 fraction of Nigella arvensis. Highest ACE-inhibitory activity of hydrolyzed protein fractions was found at 4h via pepsin hydrolysis in globulin fraction of Nigella damascena. Highest antioxidant activities of hydrolyzed protein fractions were found in glutelin-2 for both species. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel method for purification of the endogenously expressed fission yeast Set2 complex.

PubMed

Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

2014-05-01

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.

A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding

PubMed Central

Khan, Hanif M.; He, Tao; Fuglebakk, Edvin; Grauffel, Cédric; Yang, Boqian; Roberts, Mary F.; Gershenson, Anne; Reuter, Nathalie

2016-01-01

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought. PMID:27028646

Biologically important compounds in synfuels processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, B R; Ho, C; Griest, W H

1980-01-01

Crude products, by-products and wastes from synfuel processes contain a broad spectrum of chemical compounds - many of which are active in biological systems. Discerning which compound classes are most important is necessary in order to establish effective control over release or exposure. Polycyclic aromatic hydrocarbons (PAH), multialkylated PAH, primary aromatic amines and N-heterocyclic PAH are significant contributors to the overall mutagenic activities of a large number of materials examined. Ames test data show that the basic, primary aromatic amine fraction is the most active. PAHs, multialkylated PAHs and N-heterocyclic PAHs are all components of the neutral fraction. In nearlymore » all cases, the neutral fractions contribute the largest portion of the mutagenic activity, while the basic primary aromatic amine fractions have the highest specific activity. Neutral fractions are usually the largest (wt %) whereas the total basic fractions are small by comparison; thus, the overall greater contribution of the neutral fraction to the mutagenic activity of most samples. Biologically active constituents are isolated in preparative scale amounts from complex mixtures utilizing combinations of liquid-liquid extraction and various liquid chromatographic column-eluant combinations. Fractions are characterized using a combination of spectroscopic techniques and gas chromatography/mass spectrometry.« less

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

Unbiased and targeted mass spectrometry for the HDL proteome.

PubMed

Singh, Sasha A; Aikawa, Masanori

2017-02-01

Mass spectrometry is an ever evolving technology that is equipped with a variety of tools for protein research. Some lipoprotein studies, especially those pertaining to HDL biology, have been exploiting the versatility of mass spectrometry to understand HDL function through its proteome. Despite the role of mass spectrometry in advancing research as a whole, however, the technology remains obscure to those without hands on experience, but still wishing to understand it. In this review, we walk the reader through the coevolution of common mass spectrometry workflows and HDL research, starting from the basic unbiased mass spectrometry methods used to profile the HDL proteome to the most recent targeted methods that have enabled an unprecedented view of HDL metabolism. Unbiased global proteomics have demonstrated that the HDL proteome is organized into subgroups across the HDL size fractions providing further evidence that HDL functional heterogeneity is in part governed by its varying protein constituents. Parallel reaction monitoring, a novel targeted mass spectrometry method, was used to monitor the metabolism of HDL apolipoproteins in humans and revealed that apolipoproteins contained within the same HDL size fraction exhibit diverse metabolic properties. Mass spectrometry provides a variety of tools and strategies to facilitate understanding, through its proteins, the complex biology of HDL.

Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hong; Yang, Yanling; Li, Yuxin

2015-02-06

Development of high resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse phase column (100 μm x 150 cm) coupled with Q Exactive MS. The column was capable of achieving a peak capacity of approximately 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was about 6 micrograms of peptides, although the column allowed loading as many as 20 micrograms. Gas phasemore » fractionation of peptide ions further increased the number of peptide identification by ~10%. Moreover, the combination of basic pH LC pre-fractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a postmortem brain sample of Alzheimer’s disease. As deep RNA sequencing of the same specimen suggested that ~16,000 genes were expressed, current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.« less

Methods to recover value-added co-products from dry grind processing of grains into fuel ethanol

USDA-ARS?s Scientific Manuscript database

Three methods were described to fractionate condensed distillers solubles (CDS) into several new co-products, including a protein-mineral fraction and a glycerol fraction by a chemical method; a protein fraction, an oil fraction and a glycerol-mineral fraction by a physical method; or a protein frac...

Emulsifying and Foaming Properties of Different Protein Fractions Obtained from a Novel Lupin Variety AluProt-CGNA(®) (Lupinus luteus).

PubMed

Burgos-Díaz, César; Piornos, José A; Wandersleben, Traudy; Ogura, Takahiro; Hernández, Xaviera; Rubilar, Mónica

2016-07-01

The use of vegetable proteins as food ingredient is becoming increasingly important due to their high versatility and environmental acceptability. This work describes a chemical characterization and techno-functional properties (emulsifying and foaming properties) of 3 protein fractions obtained from a protein-rich novel lupin variety, AluProt-CGNA(®) . This nongenetically modified variety have a great protein content in dehulled seeds (60.6 g protein/100 g, dry matter), which is higher than soybean and other lupin varieties. A simple procedure was utilized to obtain 3 different fractions by using alkali solubilization and isoelectric precipitation. Fractions 1 and 3 were mainly composed of protein and polysaccharides (NNE), whereas fraction 2 was mainly composed by protein (97%, w/w). Fraction 3 presented interesting and potential foaming properties in comparison to the other fractions evaluated in the study. Besides, its solubility, foaming and emulsifying capacity were practically not affected by pH variations. The 3 fractions also presented good emulsion stability, reaching values above a 95%. SDS-PAGE showed that fractions 1 and 2 contained mainly conglutin α, β, and δ, but in different ratios, whereas fraction 3 contained mainly conglutin γ and albumins. The results of this work will provide better understanding for the utilization of each protein fractions as potential ingredients in food industry. © 2016 Institute of Food Technologists®

In vitro anthelmintic effects of Spigelia anthelmia protein fractions against Haemonchus contortus.

PubMed

Araújo, Sandra Alves; Soares, Alexandra Martins Dos Santos; Silva, Carolina Rocha; Almeida Júnior, Eduardo Bezerra; Rocha, Cláudia Quintino; Ferreira, André Teixeira da Silva; Perales, Jonas; Costa-Júnior, Livio M

2017-01-01

Gastrointestinal nematodes are a significant concern for animal health and well-being, and anthelmintic treatment is mainly performed through the use of chemical products. However, bioactive compounds produced by plants have shown promise for development as novel anthelmintics. The aim of this study is to assess the anthelmintic activity of protein fractions from Spigelia anthelmia on the gastrointestinal nematode Haemonchus contortus. Plant parts were separated into leaves, stems and roots, washed with distilled water, freeze-dried and ground into a fine powder. Protein extraction was performed with sodium phosphate buffer (75 mM, pH 7.0). The extract was fractionated using ammonium sulfate (0-90%) and extensively dialyzed. The resulting fractions were named LPF (leaf protein fraction), SPF (stem protein fraction) and RPF (root protein fraction), and the protein contents and activities of the fractions were analyzed. H. contortus egg hatching (EHA), larval exsheathment inhibition (LEIA) and larval migration inhibition (LMIA) assays were performed. Proteomic analysis was conducted, and high-performance liquid chromatography (HPLC) chromatographic profiles of the fractions were established to identify proteins and possible secondary metabolites. S. anthelmia fractions inhibited H. contortus egg hatching, with LPF having the most potent effects (EC50 0.17 mg mL-1). During LEIA, SPF presented greater efficiency than the other fractions (EC50 0.25 mg mL-1). According to LMIA, the fractions from roots, stems and leaves also reduced the number of larvae, with EC50 values of 0.11, 0.14 and 0.21 mg mL-1, respectively. Protein analysis indicated the presence of plant defense proteins in the S. anthelmia fractions, including protease, protease inhibitor, chitinase and others. Conversely, secondary metabolites were absent in the S. anthemia fractions. These results suggest that S. anthelmia proteins are promising for the control of the gastrointestinal nematode H. contortus.

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

PubMed

Van Riper, Susan K; de Jong, Ebbing P; Carlis, John V; Griffin, Timothy J

2013-01-01

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

PubMed

Wagner, J M; Franco, M; Kephart, G M; Gleich, G J

1998-07-01

Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

Mutagenicity of aerosols from the oxidative thermal decomposition of rigid polyurethane foam.

PubMed

Zitting, A; Falck, K; Skyttä, E

1980-01-01

The aerosol fraction of the oxidative thermal decomposition products (700 degrees C) of rigid polyurethane foam was collected on glass fiber filters and fractionated into either-soluble neutral, acidic, and basic fractions and water-soluble compounds. The fractions showed mutagenic activity in a bacterial fluctuation test with Salmonella typhimurium TA98 or Escherichia coli CM891 as the tester strains. All the fractions induced mutations in both strains after metabolic activation with rat liver S-9 mix. The basic and the water-soluble fractions were mutagenic for S. typhimurium TA 98 even without activation. Thin-layer chromatography showed the presence of several primary aromatic amines in the aerosol. Polycyclic aromatic hydrocarbons were not detected by glass capillary gas chromatogaphy.

21 CFR 640.90 - Plasma Protein Fraction (Human).

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

21 CFR 640.90 - Plasma Protein Fraction (Human).

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

21 CFR 640.90 - Plasma Protein Fraction (Human).

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Plasma Protein Fraction (Human). 640.90 Section 640...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

21 CFR 640.90 - Plasma Protein Fraction (Human).

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

21 CFR 640.90 - Plasma Protein Fraction (Human).

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

Glucanases and Chitinases as Causal Agents in the Protection of Acacia Extrafloral Nectar from Infestation by Phytopathogens1[W][OA

PubMed Central

González-Teuber, Marcia; Pozo, María J.; Muck, Alexander; Svatos, Ales; Adame-Álvarez, Rosa M.; Heil, Martin

2010-01-01

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii × Nicotiana sanderae) contains “nectarins,” proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function. PMID:20023149

Comparative proteomics of matrix fractions between pimpled and normal chicken eggshells.

PubMed

Liu, Zhangguo; Song, Lingzi; Lu, Lizhi; Zhang, Xianfu; Zhang, Fuming; Wang, Kehua; Linhardt, Robert J

2017-09-07

Eggshell matrix can be dissociated into three matrix fractions: acid-insoluble matrix (M1), water-insoluble matrix (M2) and acid-water facultative-soluble matrix (M3). Matrix fractions from pimpled and normal eggshells were compared using label-free proteomic method to understand the differences among three matrix fractions and the proteins involved with eggshell quality. A total of 738 and 600 proteins were identified in the pimpled and normal calcified eggshells, respectively. Both eggshells showed a combined proteomic inventory of 769 proteins. In the same type of eggshell, a high similarity was present in the proteomes of three matrix fractions. These triply overlapped common proteins formed the predominant contributor to proteomic abundance in the matrix fractions. In each matrix fraction and between both eggshell models, normal and pimpled eggshells, a majority of the proteomes of the fractions were commonly observed. Forty-two common major proteins (iBAQ-derived abundance ≥0.095% of proteomic abundance) were identified throughout the three matrix fractions and these proteins might act as backbone constituents in chicken eggshell matrix. Finally, using 1.75-fold as up-regulated and using 0.57-fold as down-regulated cutoff values, twenty-five differential major proteins were screened and they all negatively influence and none showed any effect on eggshell quality. Overall, we uncovered the characteristics of proteomics of three eggshell matrix fractions and identified candidate proteins influencing eggshell quality. The next research on differential proteins will uncover the potential mechanisms underlying how proteins affect eggshell quality. It was reported that the proteins in an eggshell can be divided into insoluble and soluble proteins. The insoluble proteins are thought to be an inter-mineral matrix and acts as a structural framework, while the soluble proteins are thought as intra-mineral matrix that are embedded within the crystal during calcification. However, the difference between matrix fractions is unknown. Cross-analysis of proteomic data of three matrix fractions from the same type of eggshell, uncovered triply overlapped common proteins formed the predominant contributor to proteomic abundance of any matrix fraction, and we suggested that abundance variance of some common proteins between the three matrix fractions might be an important cause of their solubility differences. Moreover, eggshell is formed in hen's uterus, and uterus tend to be considered as unique organ determining eggshell quality. By cross-analysis on proteomic data of three matrix fractions between two eggshell models, normal and pimpled eggshells, the differential proteins were screened as candidates influencing eggshell quality. And we suggested that the liver and spleen or lymphocytes might be the major organs influencing eggshell quality, because the most promising candidates are almost blood and non-collagenous proteins, and originated from above organs. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation".

PubMed

Cowden, R R; Rasch, E M; Curtis, S K

1976-08-12

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

PubMed Central

Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

2012-01-01

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

Effects of gravity on contractile proteins

NASA Technical Reports Server (NTRS)

Henney, H. R., Jr.

1979-01-01

A method was established for the isolation and purification of nuclei in high yield from the microplasmodia of Physarum flavicomum. Purified nuclei were resistant to breakage by methods commonly employed for isolated plant and animal nuclei. Several methods for the extraction of nuclear protein were compared. Incubation of nuclear lysates with either 2 M NaCl, with or without 5 M urea, or 1 M CaCl2 resulted in the extraction of nuclear action together with histones. The histones were chemically fractionated into the 5 basic groups common to other eucaryotic tissue. Amino acid analyses of the total histone were also performed. Nuclear actin was found to have a molecular weight of 41,000 ? 4,000 daltons as determined by SDS polyacrylamide gel electrophoresis. The amino acid composition of the nuclear action was established.

Mining the human plasma proteome with three-dimensional strategies by high-resolution Quadrupole Orbitrap Mass Spectrometry.

PubMed

Zhao, Yan; Chang, Cheng; Qin, Peibin; Cao, Qichen; Tian, Fang; Jiang, Jing; Li, Xianyu; Yu, Wenfeng; Zhu, Yunping; He, Fuchu; Ying, Wantao; Qian, Xiaohong

2016-01-21

Human plasma is a readily available clinical sample that reflects the status of the body in normal physiological and disease states. Although the wide dynamic range and immense complexity of plasma proteins are obstacles, comprehensive proteomic analysis of human plasma is necessary for biomarker discovery and further verification. Various methods such as immunodepletion, protein equalization and hyper fractionation have been applied to reduce the influence of high-abundance proteins (HAPs) and to reduce the high level of complexity. However, the depth at which the human plasma proteome has been explored in a relatively short time frame has been limited, which impedes the transfer of proteomic techniques to clinical research. Development of an optimal strategy is expected to improve the efficiency of human plasma proteome profiling. Here, five three-dimensional strategies combining HAP depletion (the 1st dimension) and protein fractionation (the 2nd dimension), followed by LC-MS/MS analysis (the 3rd dimension) were developed and compared for human plasma proteome profiling. Pros and cons of the five strategies are discussed for two issues: HAP depletion and complexity reduction. Strategies A and B used proteome equalization and tandem Seppro IgY14 immunodepletion, respectively, as the first dimension. Proteome equalization (strategy A) was biased toward the enrichment of basic and low-molecular weight proteins and had limited ability to enrich low-abundance proteins. By tandem removal of HAPs (strategy B), the efficiency of HAP depletion was significantly increased, whereas more off-target proteins were subtracted simultaneously. In the comparison of complexity reduction, strategy D involved a deglycosylation step before high-pH RPLC separation. However, the increase in sequence coverage did not increase the protein number as expected. Strategy E introduced SDS-PAGE separation of proteins, and the results showed oversampling of HAPs and identification of fewer proteins. Strategy C combined single Seppro IgY14 immunodepletion, high-pH RPLC fractionation and LC-MS/MS analysis. It generated the largest dataset, containing 1544 plasma protein groups and 258 newly identified proteins in a 30-h-machine-time analysis, making it the optimum three-dimensional strategy in our study. Further analysis of the integrated data from the five strategies showed identical distribution patterns in terms of sequence features and GO functional analysis with the 1929-plasma-protein dataset, further supporting the reliability of our plasma protein identifications. The characterization of 20 cytokines in the concentration range from sub-nanograms/milliliter to micrograms/milliliter demonstrated the sensitivity of the current strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

PubMed

Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

2017-06-01

Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds (like Boc-Gln-Ala-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC), and proteins (azocasein and azoalbumin), suggesting a specific organization of their catalytic residues. All forms are completely inhibited by specific cysteine and cysteine/serine protease inhibitors, but not by specific serine and aspartic protease inhibitors, with the sole exception of pepstatin A that significantly affects acidic bromelain forms 1 and 2. For all eight protease forms, inhibition is also observed with 1,10-phenanthrolin, a metalloprotease inhibitor. Metal ions (i.e. Mn 2+ , Mg 2+ and Ca 2+ ) showed various effects depending on the protease under consideration, but all of them are totally inhibited in the presence of Zn 2+ . Mass spectrometry analyses revealed that all forms have a molecular mass of ca. 24 kDa, which is characteristic of enzymes belonging to the papain-like proteases family. Far-UV CD spectra analysis further supported this analysis. Interestingly, secondary structure calculation proves to be highly reproducible for all cysteine proteases of the papain family tested so far (this work; see also Azarkan et al., 2011; Baeyens-Volant et al., 2015) and thus can be used as a test for rapid identification of the classical papain fold. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphotyrosine recognition domains: the typical, the atypical and the versatile

PubMed Central

2012-01-01

SH2 domains are long known prominent players in the field of phosphotyrosine recognition within signaling protein networks. However, over the years they have been joined by an increasing number of other protein domain families that can, at least with some of their members, also recognise pTyr residues in a sequence-specific context. This superfamily of pTyr recognition modules, which includes substantial fractions of the PTB domains, as well as much smaller, or even single member fractions like the HYB domain, the PKCδ and PKCθ C2 domains and RKIP, represents a fascinating, medically relevant and hence intensely studied part of the cellular signaling architecture of metazoans. Protein tyrosine phosphorylation clearly serves a plethora of functions and pTyr recognition domains are used in a similarly wide range of interaction modes, which encompass, for example, partner protein switching, tandem recognition functionalities and the interaction with catalytically active protein domains. If looked upon closely enough, virtually no pTyr recognition and regulation event is an exact mirror image of another one in the same cell. Thus, the more we learn about the biology and ultrastructural details of pTyr recognition domains, the more does it become apparent that nature cleverly combines and varies a few basic principles to generate a sheer endless number of sophisticated and highly effective recognition/regulation events that are, under normal conditions, elegantly orchestrated in time and space. This knowledge is also valuable when exploring pTyr reader domains as diagnostic tools, drug targets or therapeutic reagents to combat human diseases. PMID:23134684

The fat and protein fractions of freshwater clam ( Corbicula fluminea) extract reduce serum cholesterol and enhance bile acid biosynthesis and sterol excretion in hypercholesterolaemic rats fed a high-cholesterol diet.

PubMed

Chijimatsu, Takeshi; Umeki, Miki; Okuda, Yuji; Yamada, Koji; Oda, Hiroaki; Mochizuki, Satoshi

2011-02-01

We investigated whether the fat and protein fractions of freshwater clam (Corbicula fluminea) extract (FCE) could ameliorate hypercholesterolaemia in rats fed a high-cholesterol diet. We also explored the mechanism and the components that exert the hypocholesterolaemic effect of FCE. The doses of the fat and protein fractions were equivalent to those in 30 % FCE. The fat and protein fractions of FCE, two major components of FCE, significantly reduced the serum and hepatic cholesterol levels. The fat fraction more strongly reduced serum cholesterol levels than the same level of total FCE. The excretion of faecal neutral sterols increased in rats fed the total the FCE and the fat fraction of FCE. On the other hand, faecal bile acid levels were greater in rats fed the total FCE and the fat and protein fractions of FCE than in control animals. The hepatic gene expression of ATP-binding cassette transporter G5 and cholesterol 7α-hydroxylase was up-regulated by the administration of the total FCE and both the fat and protein fractions of FCE. These results showed that the fat and protein fractions of FCE had hypocholesterolaemic properties, and that these effects were greater with the fat fraction than with the protein fraction. The present study indicates that FCE exerts its hypocholesterolaemic effects through at least two different mechanisms, including enhanced excretion of neutral sterols and up-regulated biosynthesis of bile acids.

Staircase and Fractional Part Functions

ERIC Educational Resources Information Center

Amram, Meirav; Dagan, Miriam; Ioshpe, Michael; Satianov, Pavel

2016-01-01

The staircase and fractional part functions are basic examples of real functions. They can be applied in several parts of mathematics, such as analysis, number theory, formulas for primes, and so on; in computer programming, the floor and ceiling functions are provided by a significant number of programming languages--they have some basic uses in…

Search for breast cancer biomarkers in fractionated serum samples by protein profiling with SELDI-TOF MS.

PubMed

Opstal-van Winden, Annemieke W J; Beijnen, Jos H; Loof, Arnoud; van Heerde, Waander L; Vermeulen, Roel; Peeters, Petra H M; van Gils, Carla H

2012-01-01

Many high-abundant acute phase reactants have been previously detected as potential breast cancer biomar-kers. However, they are unlikely to be specific for breast cancer. Cancer-specific biomarkers are thought to be among the lower abundant proteins. We aimed to detect lower abundant discriminating proteins by performing serum fractionation by strong anion exchange chromatography preceding protein profiling with SELDI-TOF MS. In a pilot study, we tested the different fractions resulting from fractionation, on several array types. Fraction 3 on IMAC30 and Fraction 6 on Q10 yielded the most discriminative proteins and were used for serum protein profiling of 73 incident breast cancer cases and 73 matched controls. Eight peaks showed statistically significantly different intensities between cases and controls (P⧁0.05), and had less than 10% chance to be a false-positive finding. Seven of these were tentatively identified as apolipoprotein C-II (m/z 8,909), oxidized apolipoprotein C-II (m/z 8,925), apolipoprotein C-III (m/z 8,746), fragment of coagulation factor XIIIa (m/z 3,959), heterodimer of apolipoprotein A-I and apolipoprotein A-II (m/z 45,435), hemoglobin B-chain (m/z 15,915), and post-translational modified hemoglobin (m/z 15,346). By extensive serum fractionation, we detected many more proteins than in previous studies without fractionation. However, discriminating proteins were still high abundant. Results indicate that either lower abundant proteins are less distinctive, or more rigorous fractionation and selective protein depletion, or a more sensitive assay, are needed to detect lower abundant discriminative proteins. © 2012 Wiley Periodicals, Inc.

Reactivation of Escherichia coli cells, inactivated by ultraviolet rays, with cell extracts of propionic acid bacteria: Fractionation of the extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vorob`eva, L.I.; Khodzhaev, E.Yu.; Ponomareva, G.M.

1995-01-01

Separation of Propionibacterium shermanii extract into fractions and testing them for their reactivating effect on UV-inactivated Escherichia coli AB-1157 cells showed that the activity was associated with the fraction of soluble proteins. The activity was not demonstrated in the fractions of RNA, DNA, ribosomes, or cell walls. Fractional salting out and subsequent testing of the fractions showed two active protein fractions: fraction I (20-40% of ammonium sulfate saturating concentration) and fraction II (60-80%). These fractions were separated by HPLC into seven and eight subfractions, respectively. Reactivating activity was showed in subfraction 4 (fraction I) and subfractions 5 and 6 (fractionmore » II). Electrophoresis showed five and four polypeptides in subfractions 4 and 5, respectively. Subfraction 6 (fraction II) contained one protein with a molecular mass of about 30 kDa. This protein, apparently, was responsible for the protective properties of fraction II. 9 refs., 2 figs., 4 tabs.« less

Cadmium accumulation, sub-cellular distribution and chemical forms in rice seedling in the presence of sulfur.

PubMed

Zhang, Wen; Lin, Kuangfei; Zhou, Jian; Zhang, Wei; Liu, Lili; Zhang, Qianqian

2014-01-01

Changes in cadmium (Cd) accumulation, distribution, and chemical form in rice seedling in the joint presence of different concentrations of sulfur (S) remain almost unknown. Therefore, the indoor experiments were performed to determine the accumulation, sub-cellular distribution and chemical forms of Cd under three S levels in rice seedling for the first time. The result showed that Cd accumulation in rice roots was more than in shoots. Sub-cellular distribution of Cd in rice roots and shoots indicated that the largest proportion of Cd accumulated in cell walls and soluble fractions. As S supply increased, the proportion of Cd in cell walls reduced, while it increased in the soluble fractions. The majority of Cd existed in inorganic form, and then gradually changed to organic forms that included pectates and proteins with increased S supply. The results showed that S supply significantly influenced Cd accumulation, distribution, and chemical forms, suggesting that S might provide the material for the synthesis of sulfhydryl protein and thereby affect Cd stress on plants. These observations provided a basic understanding of potential ecotoxicological effects of joint Cd and S exposure in the environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

PubMed Central

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

2015-01-01

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner. PMID:26709825

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

PubMed

Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

2015-12-19

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Proteins in Soy Might Have a Higher Role in Cancer Prevention than Previously Expected: Soybean Protein Fractions Are More Effective MMP-9 Inhibitors Than Non-Protein Fractions, Even in Cooked Seeds.

PubMed

Lima, Ana; Oliveira, Jennifer; Saúde, Filipe; Mota, Joana; Ferreira, Ricardo Boavida

2017-02-27

The search for anticancer MMP-9 inhibitors (MMPIs) in food products has become a major goal for research. MMPIs in soy have been related only to saponins and isoflavones, but recently, low specific protein fractions in soybeans were shown to reduce MMP-9 activity as well. The present work aimed at comparing the MMPI potential of protein fractions (P) and non-protein fractions (NP) isolated from soybean seeds, before and after soaking and cooking, mimicking dietary exposures. Reverse and substrate zymography, as well as a fluoregenic DQ gelatin assay were used to evaluate MMP-9 activities. Colon cancer cell migration and proliferation was also tested in HT29 cells. Regarding MMP-9 inhibition, proteins in soy presented IC 50 values 100 times lower than non-protein extracts, and remained active after cooking, suggesting that proteins may be more effective MMP-9 inhibitors than non-protein compounds. Using the determined IC 50 concentrations, NP fractions were able to induce higher inhibitions of HT29 cell migration and proliferation, but not through MMP-9 inhibition, whilst protein fractions were shown to specifically inhibit MMP-9 activity. Overall, our results show that protein fractions in soybeans might have a higher role in soy-related cancer prevention as MMPIs than previously expected. Being nontoxic and active at lower concentrations, the discovery of these heat-resistant specific MMPI proteins in soy can be of significant importance for cancer preventive diets, particularly considering the increasing use of soy proteins in food products and the controversy around isoflavones amongst consumers.

A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.

PubMed

Bollineni, Ravi Chand; Guldvik, Ingrid J; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian G; Thiede, Bernd

2015-12-21

Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

PubMed

Poulin, Patrick; Burczynski, Frank J; Haddad, Sami

2016-02-01

A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations (IVIVEs) of toxicity (efficacy) at the cellular level from data generated in cell assays. Overall, this present study may help to optimize the human dose prediction in preclinical and clinical studies, while prescribing drugs with narrow therapeutic windows that are highly bound to extracellular proteins and/or highly ionized at the physiological pH. This may facilitate building a more accurate safety (efficacy) profile for such drugs. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Potential of Extracted Locusta Migratoria Protein Fractions as Value-Added Ingredients.

PubMed

Clarkson, Claudia; Mirosa, Miranda; Birch, John

2018-02-09

Although locusts can be sustainably produced and are nutrient rich, the thought of eating them can be hard to swallow for many consumers. This paper aims to investigate the nutritional composition of Locusta migratoria , including the properties of extracted locust protein, contributing to limited literature and product development opportunities for industry. Locusts sourced from Dunedin, New Zealand, contained a high amount of protein (50.79% dry weight) and fat (34.93%), which contained high amounts of omega-3 (15.64%), creating a desirably low omega-3/omega-6 ratio of 0.57. Three protein fractions including; insoluble locust fraction, soluble locust fraction, and a supernatant fraction were recovered following alkali isoelectric precipitation methodology. Initially, proteins were solubilised at pH 10 then precipitated out at the isoelectric point (pH 4). All fractions had significantly higher protein contents compared with the whole locust. The insoluble protein fraction represented 37.76% of the dry weight of protein recovered and was much lighter in colour and greener compared to other fractions. It also had the highest water and oil holding capacity of 5.17 mL/g and 7.31 mL/g, possibly due to larger particle size. The high supernatant yield (56.60%) and low soluble protein yield (9.83%) was unexpected and could be a result of experimental pH conditions chosen.

Potential of Extracted Locusta Migratoria Protein Fractions as Value-Added Ingredients

PubMed Central

Birch, John

2018-01-01

Although locusts can be sustainably produced and are nutrient rich, the thought of eating them can be hard to swallow for many consumers. This paper aims to investigate the nutritional composition of Locusta migratoria, including the properties of extracted locust protein, contributing to limited literature and product development opportunities for industry. Locusts sourced from Dunedin, New Zealand, contained a high amount of protein (50.79% dry weight) and fat (34.93%), which contained high amounts of omega-3 (15.64%), creating a desirably low omega-3/omega-6 ratio of 0.57. Three protein fractions including; insoluble locust fraction, soluble locust fraction, and a supernatant fraction were recovered following alkali isoelectric precipitation methodology. Initially, proteins were solubilised at pH 10 then precipitated out at the isoelectric point (pH 4). All fractions had significantly higher protein contents compared with the whole locust. The insoluble protein fraction represented 37.76% of the dry weight of protein recovered and was much lighter in colour and greener compared to other fractions. It also had the highest water and oil holding capacity of 5.17 mL/g and 7.31 mL/g, possibly due to larger particle size. The high supernatant yield (56.60%) and low soluble protein yield (9.83%) was unexpected and could be a result of experimental pH conditions chosen. PMID:29425143

Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched alpha-lactalbumin and beta-lactoglobulin food ingredients

USDA-ARS?s Scientific Manuscript database

A potentially economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (SCO2) as an acid to produce enriched fractions of alpha-lactalbumin (a-LA) and beta-lactoglobulin (b-LG) from whey protein isolate. To prepare the fractions, so...

N-glycans in liver-secreted and immunoglogulin-derived protein fractions

PubMed Central

Bekesova, S.; Kosti, O.; Chandler, K.B.; Wu, J.; Madej, H.L.; Brown, K.C.; Simonyan, V.; Goldman, R.

2013-01-01

N-glycosylation of proteins provides a rich source of information on liver disease progression because majority of serum glycoproteins, with the exception of immunoglobulins, are secreted by the liver. In this report, we present results of an optimized workflow for MALDI-TOF analysis of permethylated N-glycans detached from serum proteins and separated into liver secreted and immunoglobulin fractions. We have compared relative intensities of N-glycans in 23 healthy controls and 23 cirrhosis patients. We were able to detect 82 N-glycans associated primarily with liver secreted glycoproteins, 54 N-glycans in the protein G bound fraction and 52 N-glycans in the fraction bound to protein A. The N-glycan composition of the fractions differed substantially, independent of liver disease. The relative abundance of approximately 53% N-glycans in all fractions was significantly altered in the cirrhotic liver. The removal of immunoglobulins allowed detection of an increase in a series of high mannose and hybrid N-glycans associated with the liver secreted protein fraction. PMID:22326963

Initialized Fractional Calculus

NASA Technical Reports Server (NTRS)

Lorenzo, Carl F.; Hartley, Tom T.

2000-01-01

This paper demonstrates the need for a nonconstant initialization for the fractional calculus and establishes a basic definition set for the initialized fractional differintegral. This definition set allows the formalization of an initialized fractional calculus. Two basis calculi are considered; the Riemann-Liouville and the Grunwald fractional calculi. Two forms of initialization, terminal and side are developed.

STOCHASTIC INTEGRATION FOR TEMPERED FRACTIONAL BROWNIAN MOTION.

PubMed

Meerschaert, Mark M; Sabzikar, Farzad

2014-07-01

Tempered fractional Brownian motion is obtained when the power law kernel in the moving average representation of a fractional Brownian motion is multiplied by an exponential tempering factor. This paper develops the theory of stochastic integrals for tempered fractional Brownian motion. Along the way, we develop some basic results on tempered fractional calculus.

The Effects of the "Fraction Ruler" Manipulative for Teaching Computation of Fractions

ERIC Educational Resources Information Center

Schiller, Diane Profita

1977-01-01

Explores the hypothesis that students in the fourth, fifth and sixth grade who were exposed to the "fraction ruler" as a manipulative for exploring basic fraction operations would perform more successfully in addition, subtraction and multiplication problems than students taught fraction operations by the traditional method. (Author/RK)

Generalized modeling of the fractional-order memcapacitor and its character analysis

NASA Astrophysics Data System (ADS)

Guo, Zhang; Si, Gangquan; Diao, Lijie; Jia, Lixin; Zhang, Yanbin

2018-06-01

Memcapacitor is a new type of memory device generalized from the memristor. This paper proposes a generalized fractional-order memcapacitor model by introducing the fractional calculus into the model. The generalized formulas are studied and the two fractional-order parameter α, β are introduced where α mostly affects the fractional calculus value of charge q within the generalized Ohm's law and β generalizes the state equation which simulates the physical mechanism of a memcapacitor into the fractional sense. This model will be reduced to the conventional memcapacitor as α = 1 , β = 0 and to the conventional memristor as α = 0 , β = 1 . Then the numerical analysis of the fractional-order memcapacitor is studied. And the characteristics and output behaviors of the fractional-order memcapacitor applied with sinusoidal charge are derived. The analysis results have shown that there are four basic v - q and v - i curve patterns when the fractional order α, β respectively equal to 0 or 1, moreover all v - q and v - i curves of the other fractional-order models are transition curves between the four basic patterns.

[Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 11. Incorporation of 15N in the tissues and the amino acids of the muscles].

PubMed

Gruhn, K; Zander, R

1989-03-01

Over a period of 4 days 12 colostomized laying hens daily received 36 g 15N labelled wheat with 15N excess (15N') of 14.37 atom-% together with a conventional feed mixture for laying hens. The labelling of the lysine N in the wheat was 13.58 atom-%, that of histidine N 14.38 and that of arginine 15N' 13.63 atom-% 15N'. Three hens each were butchered 12, 36, 60 and 108 h after the last 15N' feeding. The first three hens did not receive any feed before being butchered. The following three hens each received the unlabelled feed ration for another 1, 2 or 4 days resp. after the main period until they were butchered. The total of skeleton muscles, the heart and the stomach muscle (without inner skin) of each hen were combined into one sample, cut thinly, drenched with fluid nitrogen and pulverized. N, 15N' and the basic and non-basic amino acids as well as their 15N' were determined in the individual samples. In contrast to the organs, the proteins in the muscle tissue have a long half life so that a slight decrease of atom-% 15N' in the muscles could only be detected after 108 h. The 14N and 15N' quota of the non-basic amino acids in the total nitrogen of the muscles is 50%. The 14N quota of the basic amino acids is 30% and the 15N' quota only 22.5% in the total muscle N. The heavy nitrogen of the free lysine in the TCA soluble N fraction is hardly detectable 36 h and 60 h after the last 15N' supply and not at all after 108 h. In contrast to this, the other two free basic amino acids remain significantly higher labelled in dependence on the last butchering time.

Methods to recover value-added coproducts from dry grind processing of grains into fuel ethanol.

PubMed

Liu, Keshun; Barrows, Frederic T

2013-07-31

Three methods are described to fractionate condensed distillers solubles (CDS) into several new coproducts, including a protein-mineral fraction and a glycerol fraction by a chemical method; a protein fraction, an oil fraction and a glycerol-mineral fraction by a physical method; or a protein fraction, an oil fraction, a mineral fraction, and a glycerol fraction by a physicochemical method. Processing factors (ethanol concentration and centrifuge force) were also investigated. Results show that the three methods separated CDS into different fractions, with each fraction enriched with one or more of the five components (protein, oil, ash, glycerol and other carbohydrates) and thus having different targeted end uses. Furthermore, because glycerol, a hygroscopic substance, was mostly shifted to the glycerol or glycerol-mineral fraction, the other fractions had much faster moisture reduction rates than CDS upon drying in a forced air oven at 60 °C. Thus, these methods could effectively solve the dewatering problem of CDS, allowing elimination of the current industrial practice of blending distiller wet grains with CDS for drying together and production of distiller dried grains as a standalone coproduct in addition to a few new fractions.

Is myelin basic protein a potential biomarker of brain cancer?

PubMed

Zavialova, M G; Shevchenko, V E; Nikolaev, E N; Zgoda, V G

2017-08-01

Myelin basic protein is a potential biomarker for the central nervous system diseases in which the myelin sheath is destroyed. Using pseudo-selected reaction monitoring and the method of standard additions, we have measured the myelin basic protein level in the cerebrospinal fluid of patients with neurotrauma (n = 6), chronic neurodegenerative diseases (n = 2) and brain cancer (n = 5). Myelin basic protein was detected only in four out of five cerebrospinal fluid samples of patients with brain cancer. The cerebrospinal fluid myelin basic protein level ranged from 3.7 to 8.8 ng ml -1 . We suggest that monitoring of myelin basic protein in cerebrospinal fluid can serve as a diagnostic test for the brain cancer.

Distinct effects of boar seminal plasma fractions exhibiting different protein profiles on the functionality of highly diluted boar spermatozoa.

PubMed

García, E M; Calvete, J J; Sanz, L; Roca, J; Martínez, E A; Vázquez, J M

2009-04-01

The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 x 10(6) spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4-6 contained low numbers of spermatozoa (<500 x 10(6)/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1-3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1-3 appear to promote sperm survival, whereas fractions 4-6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thangavelu, Pulari U.; Gupta, Vipul; Dixit, Narendra M., E-mail: narendra@chemeng.iisc.ernet.in

The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G–Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ∼0.8. The transition was triggered bymore » A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R{sub 0}, below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G–Vif axis. - Highlights: • We perform simulations and mathematical modeling of the role of APOBEC3G in suppressing HIV-1 infection. • In three distinct ways, we estimate that when over 80% of progeny virions carry APOBEC3G, productive HIV-1 infection would be suppressed. • Our estimate of this critical fraction presents quantitative guidelines for strategies targeting the APOBEC3G–Vif axis.« less

CARd-3D: Carbon Distribution in 3D Structure Program for Globular Proteins

PubMed Central

Ekambaram, Rajasekaran; Kannaiyan, Akila; Marimuthu, Vijayasarathy; Swaminathan, Vinobha Chinnaiah; Renganathan, Senthil; Perumal, Ananda Gopu

2014-01-01

Spatial arrangement of carbon in protein structure is analyzed here. Particularly, the carbon fractions around individual atoms are compared. It is hoped that it follows the principle of 31.45% carbon around individual atoms. The results reveal that globular protein's atoms follow this principle. A comparative study on monomer versus dimer reveal that carbon is better distributed in dimeric form than in its monomeric form. Similar study on solid versus liquid structures reveals that the liquid (NMR) structure has better carbon distribution over the corresponding solid (X-Ray) structure. The carbon fraction distributions in fiber and toxin protein are compared. Fiber proteins follow the principle of carbon fraction distribution. At the same time it has another broad spectrum of carbon distribution than in globular proteins. The toxin protein follows an abnormal carbon fraction distribution. The carbon fraction distribution plays an important role in deciding the structure and shape of proteins. It is hoped to help in understanding the protein folding and function. PMID:24748753

Characterization of pea (Pisum sativum) seed protein fractions.

PubMed

Rubio, Luis A; Pérez, Alicia; Ruiz, Raquel; Guzmán, M Ángeles; Aranda-Olmedo, Isabel; Clemente, Alfonso

2014-01-30

Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins. © 2013 Society of Chemical Industry.

Proteomic comparison of two fractions derived from the transsynaptic scaffold.

PubMed

Phillips, Greg R; Florens, Laurence; Tanaka, Hidekazu; Khaing, Zin Z; Fidler, Lazar; Yates, John R; Colman, David R

2005-09-15

A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF. (c) 2005 Wiley-Liss, Inc.

Proteomic Analysis of Altered Extracellular Matrix Turnover in Bleomycin-induced Pulmonary Fibrosis

PubMed Central

Decaris, Martin L.; Gatmaitan, Michelle; FlorCruz, Simplicia; Luo, Flora; Li, Kelvin; Holmes, William E.; Hellerstein, Marc K.; Turner, Scott M.; Emson, Claire L.

2014-01-01

Fibrotic disease is characterized by the pathological accumulation of extracellular matrix (ECM) proteins. Surprisingly, very little is known about the synthesis and degradation rates of the many proteins and proteoglycans that constitute healthy or pathological extracellular matrix. A comprehensive understanding of altered ECM protein synthesis and degradation during the onset and progression of fibrotic disease would be immensely valuable. We have developed a dynamic proteomics platform that quantifies the fractional synthesis rates of large numbers of proteins via stable isotope labeling and LC/MS-based mass isotopomer analysis. Here, we present the first broad analysis of ECM protein kinetics during the onset of experimental pulmonary fibrosis. Mice were labeled with heavy water for up to 21 days following the induction of lung fibrosis with bleomycin. Lung tissue was subjected to sequential protein extraction to fractionate cellular, guanidine-soluble ECM proteins and residual insoluble ECM proteins. Fractional synthesis rates were calculated for 34 ECM proteins or protein subunits, including collagens, proteoglycans, and microfibrillar proteins. Overall, fractional synthesis rates of guanidine-soluble ECM proteins were faster than those of insoluble ECM proteins, suggesting that the insoluble fraction reflected older, more mature matrix components. This was confirmed through the quantitation of pyridinoline cross-links in each protein fraction. In fibrotic lung tissue, there was a significant increase in the fractional synthesis of unique sets of matrix proteins during early (pre-1 week) and late (post-1 week) fibrotic response. Furthermore, we isolated fast turnover subpopulations of several ECM proteins (e.g. type I collagen) based on guanidine solubility, allowing for accelerated detection of increased synthesis of typically slow-turnover protein populations. This establishes the presence of multiple kinetic pools of pulmonary collagen in vivo with altered turnover rates during evolving fibrosis. These data demonstrate the utility of dynamic proteomics in analyzing changes in ECM protein turnover associated with the onset and progression of fibrotic disease. PMID:24741116

Data from quantitative label free proteomics analysis of rat spleen.

PubMed

Dudekula, Khadar; Le Bihan, Thierry

2016-09-01

The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

Representing Numbers as Continued Fractions and an N-Spire. tns Document to Do Some Basic Continued Fraction Arithmetic

ERIC Educational Resources Information Center

Leinbach, L. Carl

2015-01-01

This paper illustrates a TI N-Spire .tns file created by the author for generating continued fraction representations of real numbers and doing arithmetic with them. The continued fraction representation provides an alternative to the decimal representation. The .tns file can be used as tool for studying continued fractions and their properties as…

Improved feed protein fractionation schemes for formulating rations with the cornell net carbohydrate and protein system.

PubMed

Lanzas, C; Broderick, G A; Fox, D G

2008-12-01

Adequate predictions of rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) supplies are necessary to optimize performance while minimizing losses of excess nitrogen (N). The objectives of this study were to evaluate the original Cornell Net Carbohydrate Protein System (CNCPS) protein fractionation scheme and to develop and evaluate alternatives designed to improve its adequacy in predicting RDP and RUP. The CNCPS version 5 fractionates CP into 5 fractions based on solubility in protein precipitant agents, buffers, and detergent solutions: A represents the soluble nonprotein N, B1 is the soluble true protein, B2 represents protein with intermediate rates of degradation, B3 is the CP insoluble in neutral detergent solution but soluble in acid detergent solution, and C is the unavailable N. Model predictions were evaluated with studies that measured N flow data at the omasum. The N fractionation scheme in version 5 of the CNCPS explained 78% of the variation in RDP with a root mean square prediction error (RMSPE) of 275 g/d, and 51% of the RUP variation with RMSPE of 248 g/d. Neutral detergent insoluble CP flows were overpredicted with a mean bias of 128 g/d (40% of the observed mean). The greatest improvements in the accuracy of RDP and RUP predictions were obtained with the following 2 alternative schemes. Alternative 1 used the inhibitory in vitro system to measure the fractional rate of degradation for the insoluble protein fraction in which A = nonprotein N, B1 = true soluble protein, B2 = insoluble protein, C = unavailable protein (RDP: R(2) = 0.84 and RMSPE = 167 g/d; RUP: R(2) = 0.61 and RMSPE = 209 g/d), whereas alternative 2 redefined A and B1 fractions as the non-amino-N and amino-N in the soluble fraction respectively (RDP: R(2) = 0.79 with RMSPE = 195 g/d and RUP: R(2) = 0.54 with RMSPE = 225 g/d). We concluded that implementing alternative 1 or 2 will improve the accuracy of predicting RDP and RUP within the CNCPS framework.

21 CFR 640.91 - Processing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.91 Processing. (a) Date... °C or colder. (e) Heat treatment. Heating of the final containers of Plasma Protein Fraction (Human... concentration of the product. (g) Incubation. All final containers of Plasma Protein Fraction (Human) shall be...

21 CFR 640.91 - Processing.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.91 Processing. (a) Date... °C or colder. (e) Heat treatment. Heating of the final containers of Plasma Protein Fraction (Human... concentration of the product. (g) Incubation. All final containers of Plasma Protein Fraction (Human) shall be...

21 CFR 640.91 - Processing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.91 Processing. (a) Date... °C or colder. (e) Heat treatment. Heating of the final containers of Plasma Protein Fraction (Human... concentration of the product. (g) Incubation. All final containers of Plasma Protein Fraction (Human) shall be...

21 CFR 640.91 - Processing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.91 Processing. (a) Date... °C or colder. (e) Heat treatment. Heating of the final containers of Plasma Protein Fraction (Human... concentration of the product. (g) Incubation. All final containers of Plasma Protein Fraction (Human) shall be...

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

NASA Technical Reports Server (NTRS)

Li, H.; Dauwalder, M.; Roux, S. J.

1991-01-01

Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

Lack of Dependence of the Sizes of the Mesoscopic Protein Clusters on Electrostatics.

PubMed

Vorontsova, Maria A; Chan, Ho Yin; Lubchenko, Vassiliy; Vekilov, Peter G

2015-11-03

Protein-rich clusters of steady submicron size and narrow size distribution exist in protein solutions in apparent violation of the classical laws of phase equilibrium. Even though they contain a minor fraction of the total protein, evidence suggests that they may serve as essential precursors for the nucleation of ordered solids such as crystals, sickle-cell hemoglobin polymers, and amyloid fibrils. The cluster formation mechanism remains elusive. We use the highly basic protein lysozyme at nearly neutral and lower pH as a model and explore the response of the cluster population to the electrostatic forces, which govern numerous biophysical phenomena, including crystallization and fibrillization. We tune the strength of intermolecular electrostatic forces by varying the solution ionic strength I and pH and find that despite the weaker repulsion at higher I and pH, the cluster size remains constant. Cluster responses to the presence of urea and ethanol demonstrate that cluster formation is controlled by hydrophobic interactions between the peptide backbones, exposed to the solvent after partial protein unfolding that may lead to transient protein oligomers. These findings reveal that the mechanism of the mesoscopic clusters is fundamentally different from those underlying the two main classes of ordered protein solid phases, crystals and amyloid fibrils, and partial unfolding of the protein chain may play a significant role. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Evaluation of protein fractionation systems used in formulating rations for dairy cattle.

PubMed

Lanzas, C; Tedeschi, L O; Seo, S; Fox, D G

2007-01-01

Production efficiency decreases when diets are not properly balanced for protein. Sensitivity analyses of the protein fractionation schemes used by the National Research Council Nutrient Requirement of Dairy Cattle (NRC) and the Cornell Net Carbohydrate and Protein System (CNCPS) were conducted to assess the influence of the uncertainty in feed inputs and the assumptions underlying the CNCPS scheme on metabolizable protein and amino acid predictions. Monte Carlo techniques were used. Two lactating dairy cow diets with low and high protein content were developed for the analysis. A feed database provided by a commercial laboratory and published sources were used to obtain the distributions and correlations of the input variables. Spreadsheet versions of the models were used. Both models behaved similarly when variation in protein fractionation was taken into account. The maximal impact of variation on metabolizable protein from rumen-undegradable protein (RUP) was 2.5 (CNCPS) and 3.0 (NRC) kg/d of allowable milk for the low protein diet, and 3.5 (CNCPS) and 3.9 (NRC) kg/d of allowable milk for the high protein diet. The RUP flows were sensitive to ruminal degradation rates of the B protein fraction in NRC and of the B2 protein fraction in the CNCPS for protein supplements, energy concentrates, and forages. Absorbed Met and Lys flows were also sensitive to intestinal digestibility of RUP, and the CNCPS model was sensitive to acid detergent insoluble crude protein and its assumption of complete unavailability. Neither the intestinal digestibility of the RUP nor the protein degradation rates are routinely measured. Approaches need to be developed to account for their variability. Research is needed to provide better methods for measuring pool sizes and ruminal digestion rates for protein fractionation systems.

Fractionation of whey protein isolate with supercritical carbon dioxide – process modeling and cost estimation

USDA-ARS?s Scientific Manuscript database

An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of alpha-lactalbumin (alpha-La) and beta-lactoglobulin (beta-Lg) from a commercial whey protein isolate (WPI) containing 55% ...

Pilot-scale fractionation of whey proteins with supercritical CO2

USDA-ARS?s Scientific Manuscript database

A new pilot-scale process is being developed and optimized for the separation of whey proteins into two enriched, highly functional fractions that are free of contaminants. The fractionation of whey protein isolate (WPI), which contains approximately 32% alpha-lactalbumin (alpha-LA) and 61% beta-lac...

Overview of Cell Synchronization.

PubMed

Banfalvi, Gaspar

2017-01-01

The widespread interest in cell synchronization is maintained by the studies of control mechanism involved in cell cycle regulation. During the synchronization distinct subpopulations of cells are obtained representing different stages of the cell cycle. These subpopulations are then used to study regulatory mechanisms of the cycle at the level of macromolecular biosynthesis (DNA synthesis, gene expression, protein synthesis), protein phosphorylation, development of new drugs, etc. Although several synchronization methods have been described, it is of general interest that scientists get a compilation and an updated view of these synchronization techniques. This introductory chapter summarizes: (1) the basic concepts and principal criteria of cell cycle synchronizations, (2) the most frequently used synchronization methods, such as physical fractionation (flow cytometry, dielectrophoresis, cytofluorometric purification), chemical blockade, (3) synchronization of embryonic cells, (4) synchronization at low temperature, (5) comparison of cell synchrony techniques, (6) synchronization of unicellular organisms, and (7) the effect of synchronization on transfection.

The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

PubMed

Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

2014-07-15

In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural proteins in the egg-shell of the oriental garden cricket, Gryllus mitratus

PubMed Central

Kawasaki, Hiroya; Sato, Hitoshi; Suzuki, Motoko

1971-01-01

1. The egg-shell of the oriental garden cricket, Gryllus mitratus, contained at least two different types of structural protein in an approximate ratio of 5:1. The major fraction was extracted in a solvent containing dithiothreitol, EDTA and 8m-urea, and was purified to apparent homogeneity as judged by free-boundary electrophoresis and ultracentrifugation. This was designated SH-fraction and its S-carboxymethyl derivative (CM-fraction) was also prepared. The minor fraction, insoluble in the solvent, was designated insoluble residue. 2. The major fraction was a phosphoprotein, rich in serine (29.8mol% of the total amino acids) and phosphate (nearly equimolar to serine), and O-phosphoserine was identified in its partial acid hydrolysate. The content of cystine was rather low (0.9mol%) in spite of the importance of this amino acid residue in the native form of the protein. The insoluble residue contained only a small amount of phosphorus, and its amino acid composition was clearly different from the major fraction. 3. CM-fraction, a fibrous protein with an average molecular weight of 57500, behaved as a typical polyanion owing to the high content of phosphate. SH-fraction and CM-fraction were precipitable from their aqueous solutions by the addition of bivalent metal cations, and the precipitation of CM-fraction by Ca2+ and Mg2+ was studied in detail. 4. When SH-fraction was exposed to air, intermolecular disulphide linkages were formed, yielding a net-like gel that changed its volume with changes in Ca2+, Mg2+ and Na+. 5. The possible role of this protein fraction in maintaining the integrity of the egg-shell, and a comparison of its composition and properties with other egg-shell proteins and other phosphoproteins, are discussed. ImagesFig. 2.PLATE 1 PMID:5004198

Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

PubMed

Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

2012-01-01

Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

Reorganization of low-molecular-weight fraction of plasma proteins in the annual cycle of cyprinidae.

PubMed

Andreeva, A M; Lamas, N E; Serebryakova, M V; Ryabtseva, I P; Bolshakov, V V

2015-02-01

Reorganization of the low-molecular-weight fraction of cyprinid plasma was analyzed using various electrophoretic techniques (disc electrophoresis, electrophoresis in polyacrylamide concentration gradient, in polyacrylamide with urea, and in SDS-polyacrylamide). The study revealed coordinated changes in the low-molecular-weight protein fractions with seasonal dynamics and related reproductive rhythms of fishes. We used cultured species of the Cyprinidae family with sequenced genomes for the detection of these interrelations in fresh-water and anadromous cyprinid species. The common features of organization of fish low-molecular-weight plasma protein fractions made it possible to make reliable identification of their proteins. MALDI mass-spectrometry analysis revealed the presence of the same proteins (hemopexin, apolipoproteins, and serpins) in the low-molecular-weight plasma fraction in wild species and cultured species with sequenced genomes (carp, zebrafish). It is found that the proteins of the first two classes are organized as complexes made of protein oligomers. Stoichiometry of these complexes changes in concordance with the seasonal and reproductive rhythms.

Unique fluorescence and high-molecular weight characteristics of protein isolates from manuka honey (Leptospermum scoparium).

PubMed

Rückriemen, Jana; Hohmann, Christoph; Hellwig, Michael; Henle, Thomas

2017-09-01

This study compared the fluorescence properties (λ ex/em =350/450nm) and molecular size of proteins from manuka and non-manuka honey. The fluorescence characteristics of non-manuka and manuka proteins differ markedly, whereby manuka honey protein fluorescence increases with increasing methylglyoxal (MGO) content of the honey. It was concluded that manuka honey proteins are modified due to MGO-derived glycation and crosslinking reactions, thus resulting in fluorescent structures. The molecular size of honey proteins was studied using size exclusion chromatography. Manuka honey proteins contain a significantly higher amount of high molecular weight (HMW) fraction compared to non-manuka honey proteins. Moreover, HMW fraction of manuka honey proteins was stable against reducing agents such as dithiothreitol, whereas HMW fraction of non-manuka honey proteins was significantly decreased. Thus, the chemical nature of manuka honey HMW fraction is probably covalent MGO crosslinking, whereas non-manuka HMW fraction is caused by disulfide bonds. Storage of a non-manuka honey, which was artificially spiked with MGO and DHA, did not induce above mentioned fluorescence properties of proteins during 84days of storage. Hence, MGO-derived fluorescence and crosslinking of honey proteins can be useful parameters to characterize manuka honey. Copyright © 2017 Elsevier Ltd. All rights reserved.

Study of Fractions in Elementary Mathematics Textbooks from Finland and Taiwan

ERIC Educational Resources Information Center

Yang, Der-Ching

2018-01-01

This study examines the different ways in which fractions are dealt with in two elementary mathematics textbook series: Kang Hsuan (KH) in Taiwan and Laskutaito in Finland. The results showed that the total number of fraction questions in Laskutaito is higher than that in KH and that Laskutaito highlights the basic definition of fractions to help…

Polypeptide composition of fraction 1 protein of the somatic hybrid between Petunia parodii and Petunia parviflora.

PubMed

Kumar, A; Wilson, D; Cocking, E C

1981-04-01

The analysis of the subunit polypeptide composition of Fraction 1 protein provides information on the expression of both chloroplast and nuclear genomes. Fraction 1 protein, isolated from leaves of the somatic hybrid plants derived form the fusion of protoplasts of Petunia parodii and P. parviflora, was analyzed for its subunit polypeptide composition by isoelectric focusing in 8 M urea. The fraction 1 protein enzyme oligomer in the somatic hybrid plants contained small subunits resulting from the expression of both parental nuclear genomes, but probably only one of the parental large subunits, namely that of P. parodii. The relevance of such somatic hybrid material for the study of nucleocytoplasmic interrelationship is discussed, as well as the use of these fraction 1 protein isoelectric focusing patterns for the analysis of taxonomic relationships in Petunia.

SBION: A Program for Analyses of Salt-Bridges from Multiple Structure Files.

PubMed

Gupta, Parth Sarthi Sen; Mondal, Sudipta; Mondal, Buddhadev; Islam, Rifat Nawaz Ul; Banerjee, Shyamashree; Bandyopadhyay, Amal K

2014-01-01

Salt-bridge and network salt-bridge are specific electrostatic interactions that contribute to the overall stability of proteins. In hierarchical protein folding model, these interactions play crucial role in nucleation process. The advent and growth of protein structure database and its availability in public domain made an urgent need for context dependent rapid analysis of salt-bridges. While these analyses on single protein is cumbersome and time-consuming, batch analyses need efficient software for rapid topological scan of a large number of protein for extracting details on (i) fraction of salt-bridge residues (acidic and basic). (ii) Chain specific intra-molecular salt-bridges, (iii) inter-molecular salt-bridges (protein-protein interactions) in all possible binary combinations (iv) network salt-bridges and (v) secondary structure distribution of salt-bridge residues. To the best of our knowledge, such efficient software is not available in public domain. At this juncture, we have developed a program i.e. SBION which can perform all the above mentioned computations for any number of protein with any number of chain at any given distance of ion-pair. It is highly efficient, fast, error-free and user friendly. Finally we would say that our SBION indeed possesses potential for applications in the field of structural and comparative bioinformatics studies. SBION is freely available for non-commercial/academic institutions on formal request to the corresponding author (akbanerjee@biotech.buruniv.ac.in).

Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

PubMed Central

DeHart, Caroline J.; Schweitzer, Mary H.; Thomas, Paul M.; Kelleher, Neil L.

2016-01-01

Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils. PMID:27812413

Chromatographic fractionation and molecular mass characterization of Cercidium praecox (Brea) gum.

PubMed

Castel, Virginia; Zivanovic, Svetlana; Jurat-Fuentes, Juan L; Santiago, Liliana G; Rubiolo, Amelia C; Carrara, Carlos R; Harte, Federico M

2016-10-01

Brea gum (BG) is an exudate from the Cercidium praecox tree that grows in semi-arid regions of Argentina. Some previous studies on BG have shown physicochemical characteristics and functional features similar to those of gum arabic. However, there is a need to elucidate the molecular structure of BG to understand the functionality. In this sense, BG was fractionated using hydrophobic interaction chromatography and the obtained fractions were analyzed by size exclusion chromatography. Analysis of the fractions showed that the bulk of the gum (approx. 84% of the polysaccharides) was a polysaccharide of 2.79 × 10(3)  kDa. The second major fraction (approx. 16% of the polysaccharides) was a polysaccharide-protein complex with a molecular mass of 1.92 × 10(5)  kDa. A third fraction consisted of protein species with a wide range of molecular weights. The molecular weight distribution of the protein fraction was analyzed by size exclusion chromatography. Comparison of the elution profiles of the exudates in native and reducing conditions revealed that some of the proteins were forming aggregates through disulfide bridges in native conditions. Further analysis of the protein fraction by SDS-PAGE showed proteins with molecular weight ranging from 6.5 to 66 kDa. The findings showed that BG consists of several fractions with heterogeneous chemical composition and polydisperse molecular weight distributions. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Amino acid sequence of the smaller basic protein from rat brain myelin

PubMed Central

Dunkley, Peter R.; Carnegie, Patrick R.

1974-01-01

1. The complete amino acid sequence of the smaller basic protein from rat brain myelin was determined. This protein differs from myelin basic proteins of other species in having a deletion of a polypeptide of 40 amino acid residues from the centre of the molecule. 2. A detailed comparison is made of the constant and variable regions in a group of myelin basic proteins from six species. 3. An arginine residue in the rat protein was found to be partially methylated. The ratio of methylated to unmethylated arginine at this position differed from that found for the human basic protein. 4. Three tryptic peptides were isolated in more than one form. The differences between the two forms of each peptide are discussed in relation to the electrophoretic heterogeneity of myelin basic proteins, which is known to occur at alkaline pH values. 5. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50029 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4141893

Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

PubMed Central

Corfield, M. C.; Fletcher, J. C.; Robson, A.

1967-01-01

1. A tryptic digest of the protein fraction U.S.3 from oxidized wool has been separated into 32 peptide fractions by cation-exchange resin chromatography. 2. Most of these fractions have been resolved into their component peptides by a combination of the techniques of cation-exchange resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid compositions of 58 of the peptides in the digest present in the largest amounts have been determined. 4. The amino acid sequences of 38 of these have been completely elucidated and those of six others partially derived. 5. These findings indicate that the parent protein in wool from which the protein fraction U.S.3 is derived has a minimum molecular weight of 74000. 6. The structures of wool proteins are discussed in the light of the peptide sequences determined, and, in particular, of those sequences in fraction U.S.3 that could not be elucidated. PMID:16742497

Inhibition of Staphylococcus aureus by crude and fractionated extract from lactic acid bacteria.

PubMed

Wong, C-B; Khoo, B-Y; Sasidharan, S; Piyawattanametha, W; Kim, S H; Khemthongcharoen, N; Ang, M-Y; Chuah, L-O; Liong, M-T

2015-03-01

Increasing levels of antibiotic resistance by Staphylococcus aureus have posed a need to search for non-antibiotic alternatives. This study aimed to assess the inhibitory effects of crude and fractionated cell-free supernatants (CFS) of locally isolated lactic acid bacteria (LAB) against a clinical strain of S. aureus. A total of 42 LAB strains were isolated and identified from fresh vegetables, fresh fruits and fermented products prior to evaluation of inhibitory activities. CFS of LAB strains exhibiting a stronger inhibitive effect against S. aureus were fractionated into crude protein, polysaccharide and lipid fractions. Crude protein fractions showed greater inhibition against S. aureus compared to polysaccharide and lipid fractions, with a more prevalent effect from Lactobacillus plantarum 8513 and L. plantarum BT8513. Crude protein, polysaccharide and lipid fractions were also characterised with glycine, mannose and oleic acid being detected as the major component of each fraction, respectively. Scanning electron microscopy revealed roughed and wrinkled membrane morphology of S. aureus upon treatment with crude protein fractions of LAB, suggesting an inhibitory effect via the destruction of cellular membrane. This research illustrated the potential application of fractionated extracts from LAB to inhibit S. aureus for use in the food and health industry.

Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

PubMed Central

Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

2005-01-01

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

21 CFR 862.1630 - Protein (fractionation) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test... body fluids. Protein fractionations are used as an aid in recognizing abnormal proteins in body fluids...

21 CFR 862.1630 - Protein (fractionation) test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test... body fluids. Protein fractionations are used as an aid in recognizing abnormal proteins in body fluids...

21 CFR 862.1630 - Protein (fractionation) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test... body fluids. Protein fractionations are used as an aid in recognizing abnormal proteins in body fluids...

21 CFR 862.1630 - Protein (fractionation) test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test... body fluids. Protein fractionations are used as an aid in recognizing abnormal proteins in body fluids...

Transduction of NeuroD2 protein induced neural cell differentiation.

PubMed

Noda, Tomohide; Kawamura, Ryuzo; Funabashi, Hisakage; Mie, Masayasu; Kobatake, Eiry

2006-11-01

NeuroD2, one of the neurospecific basic helix-loop-helix transcription factors, has the ability to induce neural differentiation in undifferentiated cells. In this paper, we show that transduction of NeuroD2 protein induced mouse neuroblastoma cell line N1E-115 into neural differentiation. NeuroD2 has two basic-rich domains, one is nuclear localization signal (NLS) and the other is basic region of basic helix-loop-helix (basic). We constructed some mutants of NeuroD2, ND2(Delta100-115) (lack of NLS), ND2(Delta123-134) (lack of basic) and ND2(Delta100-134) (lack of both NLS and basic) for transduction experiments. Using these proteins, we have shown that NLS region of NeuroD2 plays a role of protein transduction. Continuous addition of NeuroD2 protein resulted in N1E-115 cells adopting neural morphology after 4 days and Tau mRNA expression was increased. These results suggest that neural differentiation can be induced by direct addition of NeuroD2 protein.

Antitumor activity against murine lymphoma L5178Y model of proteins from cacao (Theobroma cacao L.) seeds in relation with in vitro antioxidant activity.

PubMed

Preza, Ana M; Jaramillo, María E; Puebla, Ana M; Mateos, Juan C; Hernández, Rodolfo; Lugo, Eugenia

2010-10-20

Recently, proteins and peptides have become an added value to foodstuffs due to new knowledge about its structural analyses as related to antioxidant and anticancer activity. Our goal was to evaluate if protein fractions from cacao seeds show antitumor activity on lymphoma murine L5178Y model. The antioxidant activity of these fractions was also evaluated with the aim of finding a correlation with the antitumor activity. Differential extraction of proteins from unfermented and semi-fermented-dry cacao seeds was performed and characterized by SDS-PAGE and FPLC size-exclusion chromatography. Antitumor activity was evaluated against murine lymphoma L5178Y in BALB/c mice (6 × 104 cells i.p.), with a treatment oral dose of 25 mg/kg/day of each protein fraction, over a period of 15 days. Antioxidant activity was evaluated by the ABTS+ and ORAC-FL assays. Albumin, globulin and glutelin fractions from both cacao seed type were obtained by differential solubility extraction. Glutelins were the predominant fraction. In the albumin fraction, polypeptides of 42.3 and 8.5 kDa were found in native conditions, presumably in the form of two peptide chains of 21.5 kDa each one. The globulin fraction presented polypeptides of 86 and 57 kDa in unfermented cacao seed that produced the specific-cacao aroma precursors, and after fermentation the polypeptides were of 45 and 39 kDa. The glutelin fraction presented proteins >200 kDa and globulins components <100 KDa in lesser proportion. Regarding the semifermented-dry cacao seed, it was observed that the albumin fraction showed antitumoral activity, since it caused significant decreases (p < 0.05) in the ascetic fluid volume and packed cell volume, inhibiting cell growth in 59.98 ± 13.6% at 60% of the population; while the greatest antioxidant capacity due to free radical scavenging capacity was showed by the albumin and glutelin fraction in both methods assayed. This study is the first report on the biological activity of semifermented-dry cacao protein fractions with their identification, supporting the traditional use of the plant. The albumin fraction showed antitumor and free radical scavenging capacity, however both activities were not correlated. The protein fractions could be considered as source of potential antitumor peptides.

Antitumor activity against murine lymphoma L5178Y model of proteins from cacao (Theobroma cacao L.) seeds in relation with in vitro antioxidant activity

PubMed Central

2010-01-01

Background Recently, proteins and peptides have become an added value to foodstuffs due to new knowledge about its structural analyses as related to antioxidant and anticancer activity. Our goal was to evaluate if protein fractions from cacao seeds show antitumor activity on lymphoma murine L5178Y model. The antioxidant activity of these fractions was also evaluated with the aim of finding a correlation with the antitumor activity. Methods Differential extraction of proteins from unfermented and semi-fermented-dry cacao seeds was performed and characterized by SDS-PAGE and FPLC size-exclusion chromatography. Antitumor activity was evaluated against murine lymphoma L5178Y in BALB/c mice (6 × 104 cells i.p.), with a treatment oral dose of 25 mg/kg/day of each protein fraction, over a period of 15 days. Antioxidant activity was evaluated by the ABTS+ and ORAC-FL assays. Results Albumin, globulin and glutelin fractions from both cacao seed type were obtained by differential solubility extraction. Glutelins were the predominant fraction. In the albumin fraction, polypeptides of 42.3 and 8.5 kDa were found in native conditions, presumably in the form of two peptide chains of 21.5 kDa each one. The globulin fraction presented polypeptides of 86 and 57 kDa in unfermented cacao seed that produced the specific-cacao aroma precursors, and after fermentation the polypeptides were of 45 and 39 kDa. The glutelin fraction presented proteins >200 kDa and globulins components <100 KDa in lesser proportion. Regarding the semifermented-dry cacao seed, it was observed that the albumin fraction showed antitumoral activity, since it caused significant decreases (p < 0.05) in the ascetic fluid volume and packed cell volume, inhibiting cell growth in 59.98 ± 13.6% at 60% of the population; while the greatest antioxidant capacity due to free radical scavenging capacity was showed by the albumin and glutelin fraction in both methods assayed. Conclusion This study is the first report on the biological activity of semifermented-dry cacao protein fractions with their identification, supporting the traditional use of the plant. The albumin fraction showed antitumor and free radical scavenging capacity, however both activities were not correlated. The protein fractions could be considered as source of potential antitumor peptides. PMID:20961452

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.

PubMed

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.

Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

PubMed Central

Skinner, Owen S.; Do Vale, Luis H. F.; Catherman, Adam D.; Havugimana, Pierre C.; Valle de Sousa, Marcelo; Domont, Gilberto B.; Kelleher, Neil L.; Compton, Philip D.

2016-01-01

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses. PMID:26967310

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.

PubMed

Kane, Lesley A; Yung, Christina K; Agnetti, Giulio; Neverova, Irina; Van Eyk, Jennifer E

2006-11-01

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.

Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

NASA Technical Reports Server (NTRS)

Mednieks, Maija I.; Popova, Irina A.; Grindeland, Richard E.

1991-01-01

The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

Fractionation of sheep cheese whey by a scalable method to sequentially isolate bioactive proteins.

PubMed

Pilbrow, Jodi; Bekhit, Alaa El-Din A; Carne, Alan

2016-07-15

This study reports a procedure for the simultaneous purification of glyco(caseino)macropeptide, immunoglobulin, lactoperoxidase, lactoferrin, α-lactalbumin and β-lactoglobulin from sheep cheese sweet whey, an under-utilized by-product of cheese manufacture generated by an emerging sheep dairy industry in New Zealand. These proteins have recognized value in the nutrition, biomedical and health-promoting supplements industries. A sequential fractionation procedure using economical anion and cation exchange chromatography on HiTrap resins was evaluated. The whey protein fractionation is performed under mild conditions, requires only the adjustment of pH between ion exchange chromatography steps, does not require buffer exchange and uses minimal amounts of chemicals. The purity of the whey protein fractions generated were analyzed by reversed phase-high performance liquid chromatography and the identity of the proteins was confirmed by mass spectrometry. This scalable procedure demonstrates that several proteins of recognized value can be fractionated in reasonable yield and purity from sheep cheese whey in one streamlined process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

PubMed

Acosta, Mariano; Torres, Sabier; Mariño-Repizo, Leonardo; Martinez, Luis D; Gil, Raúl A

2018-06-02

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature. Copyright © 2018 Elsevier B.V. All rights reserved.

Gel characteristics and microstructure of fish myofibrillar protein/cassava starch composites.

PubMed

Fan, Mingcong; Hu, Ting; Zhao, Siming; Xiong, Shanbai; Xie, Jing; Huang, Qilin

2017-03-01

The changes in fish myofibrillar protein/cassava starch composites in the starch fraction range from 0 to 1, with their total content maintained at 60mg/mL, were investigated in terms of textural properties, rheological behaviours, morphology, spatial distribution and protein molecular structure. The results revealed that the starch fraction of 0.5 was a critical point for the conversion of the protein matrix to starch matrix and conversion of the gel from elastic to weak. Moreover, the protein-starch synergistic effect on the storage modulus was strongest at fractions of 0.5 and 0.6, due to the formation of a semi-interpenetrating network, with more amylose from the melted starch granules interpenetrated with the protein molecules, and the absorption of water by the starch granules to concentrate the protein matrix. Additionally, no covalent interaction between the protein and starch occurred with increasing starch fraction, thus having no significant influence on the protein secondary structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity.

PubMed

Marangon, Matteo; Vincenzi, Simone; Lucchetta, Marco; Curioni, Andrea

2010-02-15

During the storage, bottled white wines can manifest haziness due to the insolubilisation of the grape proteins that may 'survive' in the fermentation process. Although the exact mechanism of this occurrence is not fully understood, proteins and tannins are considered two of the key factors involved in wine hazing, since their aggregation leads to the formation of insoluble particles. To better understand this complex interaction, proteins and tannins from the same unfined Pinot grigio wine were separated. Wine proteins were then fractionated by hydrophobic interaction chromatography (HIC). A significant correlation between hydrophobicity of the wine protein fractions and the haze formed after reacting with wine tannins was found, with the most reactive fractions revealing (by SDS-PAGE and RP-HPLC analyses) the predominant presence of thaumatin-like proteins. Moreover, the effects of both protein heating and disulfide bonds reduction (with dithiotreithol) on haze formation in the presence of tannins were assessed. These treatments generally resulted in an improved reactivity with tannins, and this phenomenon was related to both the surface hydrophobicity and composition of the protein fractions. Therefore, haze formation in wines seems to be related to hydrophobic interactions occurring among proteins and tannins. These interactions should occur on hydrophobic tannin-binding sites, whose exposition on the proteins can depend on both protein heating and reduction. Copyright 2009 Elsevier B.V. All rights reserved.

Effects of high hydrostatic pressure on the functional and rheological properties of the protein fraction extracted from pine nuts.

PubMed

Cao, Baiying; Fang, Li; Liu, Chunlei; Min, Weihong; Liu, Jingsheng

2018-01-01

High hydrostatic pressure treatments could increase the protein solubility (200 MPa), water holding capacity (400 MPa), and oil holding capacity (400 MPa) of pine nuts protein fractions, respectively. The exposed sufhydryl content for albumin was highest at 100 MPa while for other fractions it was 400 MPa, contrary for total sufhydryl content-generally it was at 100 MPa, except glutelin (400 MPa). Pine nuts protein fractions demonstrated the typical behavior of weak gels (G' > G″). After the treatments of high hydrostatic pressure the specific surface area of pine nuts protein particle was increased upon pressure, and the surface of protein became rough which increased the particle size. The functional groups of protein were found to be unchanged, but the characteristic peaks of pine nuts protein moved to a low-band displacement and the value of peaks was amplified accordingly to the pressure. The high hydrostatic pressure treatments were found to improve the functional properties of pine nuts protein isolates by enhancing the heat-induced gel strength of pine nuts protein isolates which make proteins more stretchable. These results suggest that high hydrostatic pressure treatments can increase the functional properties and alter the rheological properties of pine nuts protein fractions which will broaden its applications in food industry.

Antioxidant Activity of Coconut (Cocos nucifera L.) Protein Fractions.

PubMed

Li, Yan; Zheng, Yajun; Zhang, Yufeng; Xu, Jianguo; Gao, Gang

2018-03-20

Coconut cake is an abundant and good potential edible protein source. However, until now it has not been extensively used in the food industry. To promote its usage, the characterization, nutrition value and antioxidant activity of coconut cake protein fractions (albumin, globulin, prolamine, glutelin-1 and glutelin-2) were studied. Results revealed that all the albumin, globulin, glutelin-1 and glutelin-2 fractions showed a high nutrition value. The prolamine, glutelin-1 and glutelin-2 all exhibited good radical scavenging activity and reducing power, and the globulin and prolamine showed high ion chelating ability (89.14-80.38%). Moreover, all the fractions except glutelin-2 could effectively protect DNA against oxidative damage. Several peptides containing five to eight amino acids with antioxidant activity were also identified by LC-MS/MS from the globulin and glutelin-2 fractions. The results demonstrated that the coconut cake protein fractions have potential usages in functional foods.

Directed Shotgun Proteomics Guided by Saturated RNA-seq Identifies a Complete Expressed Prokaryotic Proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Omasits, U.; Quebatte, Maxime; Stekhoven, Daniel J.

2013-11-01

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, wemore » could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ~90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor.« less

Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome

PubMed Central

Omasits, Ulrich; Quebatte, Maxime; Stekhoven, Daniel J.; Fortes, Claudia; Roschitzki, Bernd; Robinson, Mark D.; Dehio, Christoph; Ahrens, Christian H.

2013-01-01

Prokaryotes, due to their moderate complexity, are particularly amenable to the comprehensive identification of the protein repertoire expressed under different conditions. We applied a generic strategy to identify a complete expressed prokaryotic proteome, which is based on the analysis of RNA and proteins extracted from matched samples. Saturated transcriptome profiling by RNA-seq provided an endpoint estimate of the protein-coding genes expressed under two conditions which mimic the interaction of Bartonella henselae with its mammalian host. Directed shotgun proteomics experiments were carried out on four subcellular fractions. By specifically targeting proteins which are short, basic, low abundant, and membrane localized, we could eliminate their initial underrepresentation compared to the estimated endpoint. A total of 1250 proteins were identified with an estimated false discovery rate below 1%. This represents 85% of all distinct annotated proteins and ∼90% of the expressed protein-coding genes. Genes that were detected at the transcript but not protein level, were found to be highly enriched in several genomic islands. Furthermore, genes that lacked an ortholog and a functional annotation were not detected at the protein level; these may represent examples of overprediction in genome annotations. A dramatic membrane proteome reorganization was observed, including differential regulation of autotransporters, adhesins, and hemin binding proteins. Particularly noteworthy was the complete membrane proteome coverage, which included expression of all members of the VirB/D4 type IV secretion system, a key virulence factor. PMID:23878158

Effects of carboxypeptidase B treatment and elevated temperature on recombinant monoclonal antibody charge variants in cation-exchange chromatography analysis.

PubMed

Kim, Do Gyun; Kim, Hyoung Jin; Kim, Hong-Jin

2016-10-01

Charge variants (acidic and basic) of recombinant monoclonal antibodies (Mabs) have received much attention due to their potential biological effects. C-terminal lysine variants are common in Mabs and their proportion is affected by the manufacturing process. In the present study, changes of trastuzumab charge variants brought about by carboxypeptidase B treatment and subsequent storage at 8 or 37 °C for up to 24 h were monitored by cation-exchange chromatography analysis to investigate the effects of C-terminal lysine cleavage and its subsequent reaction at 8 or 37 °C. C-terminal lysine cleavage at 8 °C reduced the fraction of basic species and had little effect on the fraction of acidic species. Analysis of individual peaks demonstrated that C-terminal lysine cleavage induced both increases and decreases in individual acidic variants, with the result that there was little overall change in the overall proportion of acidic species. It appeared that most of the basic variant Mab molecules but only a fraction of the acidic variant molecules had C-terminal lysines. Increasing the temperature to 37 °C appeared to increase the fraction of acidic species and decrease main species significantly, without a similar change in basic species. These results indicate that length of exposure to elevated temperature is a critical consideration in charge variant analysis.

[Protein fractions and their enzyme activity in the rat myocardium in a Kosmos-936 biosatellite experiment].

PubMed

Tigranian, R A; Nosova, E A; Kolchina, E V; Veresotskaia, N A; Kurkina, L M

1981-01-01

The effect of artificial gravity on protein fractions and their enzyme activity in the myocardium of rats flown on board Cosmos-936 was studied. In weightless rats the content of sarcoplasmic proteins increased at R + O and that of T fraction proteins decreased at R + 25. In centrifuged rats such changes were not seen. In centrifuged rats the enzyme activity of sarcoplasmic proteins did not alter. In weightless rats ATPase activity of myosin decreased significantly, and in centrifuged rats it remained almost unchanged.

A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl

2009-10-02

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this systemmore » using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.« less

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

PubMed Central

Schlautman, Joshua D; Rozek, Wojciech; Stetler, Robert; Mosley, R Lee; Gendelman, Howard E; Ciborowski, Pawel

2008-01-01

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. PMID:18789151

Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry

NASA Astrophysics Data System (ADS)

Choi, Sam B.; Lombard-Banek, Camille; Muñoz-LLancao, Pablo; Manzini, M. Chiara; Nemes, Peter

2018-05-01

The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An 1 to 20 μg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates 274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to 57%. From 1 ng of protein digest (<5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or 480 protein groups ( 1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest ( a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization. [Figure not available: see fulltext.

Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry

NASA Astrophysics Data System (ADS)

Choi, Sam B.; Lombard-Banek, Camille; Muñoz-LLancao, Pablo; Manzini, M. Chiara; Nemes, Peter

2017-11-01

The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An 1 to 20 μg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates 274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to 57%. From 1 ng of protein digest (<5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or 480 protein groups ( 1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest ( a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization. [Figure not available: see fulltext.

STUDY OF THE PROTEIN FRACTIONS IN THE BRAIN AFTER EXPOSURE TO X RAYS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Narepekha, O.M.

1962-01-01

A study was made of the changes in the water-soluble proteins of the brain in 10 adult rabbits exposed to totalbody radiation by means of a dose of 800 r at a rate of 16 r/min. After the exposure, the brain was cleaned of blood, meninges, and blood vessels, and was homogenized with an equal volume of normal saline. The homogenate was frozen by means of liquid nitrogen and left in the deep freeze for 24 hours. The homogenate was then melted, and centrifuged for one hour at 15,000 rpm. The supernatant liquid obtained had a protein content of 1.8more » to 2%. To increase this to the concentration of serum protein (7 to 8%), the solution was precipitated with tannin and the protein was liberated from the protein-tannate complex with caffeine. The pH adjusted to 4.7. After ten minutes, the solution was centrifuged, the supernatant liquid was discarded, and the centrifugate washed twice in normal saline and redissolved by the addition of caffeine. The solution was centrifuged for 30 minutes at 15,000 rpm, after which the centrifugate contained the protein liberated from the protein-tannate complex. The solution obtained had a protein concentration of 9 to 10%. This was then investigated by electrophoresis on agar gel in a veronal-medinal buffer (pH 8.6), at a voltage of 220 v. In the control rabbits, electrophoresis of the solution obtained in the described manner yields 9 to 11 fractions, one of which was a prealbumin fraction, the second an albumin fraction, and the others corresponded to various serum-globulin fractions. In rabbits exposed to radiation, the number of fractions increased to 13, mainly fractions corresponding to the serum- albumins. (OTS)« less

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

PubMed

Rocha, Surza L G; Neves-Ferreira, Ana G C; Trugilho, Monique R O; Angulo, Yamileth; Lomonte, Bruno; Valente, Richard H; Domont, Gilberto B; Perales, Jonas

2017-01-16

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA 2 , a few spots corresponding to acidic PLA 2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. Local tissue damage induced by myotoxic PLA 2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein. Copyright © 2016. Published by Elsevier B.V.

Developing Basic Math Skills for Marketing. Student Manual and Laboratory Guide.

ERIC Educational Resources Information Center

Klewer, Edwin D.

Field tested with students in grades 10-12, this manual is designed to teach students in marketing courses basic mathematical concepts. The instructional booklet contains seven student assignments covering the following topics: why basic mathematics is so important, whole numbers, fractions, decimals, percentages, weights and measures, and dollars…

Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion

PubMed Central

Liu, Haipeng; Yu, Jia; Qiao, Rui; Zhou, Mi; Yang, Yongtao; Zhou, Jian; Xie, Peng

2016-01-01

The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest. PMID:27832179

THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS.

PubMed

A, Güllü; S, Dede

2016-01-01

TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control's (p<0.05). In addition, it was observed that the γ-globulin levels of TQ group were lower than that of the control group's (p<0.05). From the results, it was observed that the changes of these fractions may have originated from elevation or decline synthesis, or activities of containing proteins.

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

PubMed Central

Paton, B C; Poulos, A

1988-01-01

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands. Images Fig. 1. Fig. 2. Fig. 3. PMID:3178760

Perturbations in the Urinary Exosome in Transplant Rejection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigdel, Tara K.; NG, Yolanda; Lee, Sangho

Background: Urine exosomes, vesicles exocytosed into urine by all renal epithelial cell types, occur under normal physiologic and disease states. Exosome contents may mirror disease-specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed and for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery. Methods: Urine exosomes were isolated by centrifugal filtration from mid-stream, second morning void, urine samples collected from kidney transplant recipients with and without biopsy matched acute rejection. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Uexo) underwent mass spectrometry-based quantitative proteomics analysis. Themore » proteome data were analyzed for significant differential protein abundances in acute rejection (AR). Results: Identifications of 1018 and 349 proteins, Uw and Uexo fractions, respectively, demonstrated a 279 protein overlap between the two urinary compartments with 25%(70) of overlapping proteins unique to Uexoand represented membrane bound proteins (p=9.31e-7). Of 349 urine exosomal proteins identified in transplant patients 220 were not previously identified in the normal urine exosomal fraction. Uexo proteins (11), functioning in the inflammatory / stress response, were more abundant in patients with biopsy-confirmed acute rejection, 3 of which were exclusive to Uexo. Uexo AR-specific biomarkers (8) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw. Conclusions: A rapid urinary exosome isolation method and quantitative measurement of enriched Uexo proteins was applied. Urine proteins specific to the exosomal fraction were detected either in unfractionated urine (at low abundances) or by Uexo fraction analysis. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Uexo fraction from normal healthy subjects. Uexo specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring for acute transplant rejection.« less

Rapid preparation of nuclei-depleted detergent-resistant membrane fractions suitable for proteomics analysis.

PubMed

Adam, Rosalyn M; Yang, Wei; Di Vizio, Dolores; Mukhopadhyay, Nishit K; Steen, Hanno

2008-06-05

Cholesterol-rich membrane microdomains known as lipid rafts have been implicated in diverse physiologic processes including lipid transport and signal transduction. Lipid rafts were originally defined as detergent-resistant membranes (DRMs) due to their relative insolubility in cold non-ionic detergents. Recent findings suggest that, although DRMs are not equivalent to lipid rafts, the presence of a given protein within DRMs strongly suggests its potential for raft association in vivo. Therefore, isolation of DRMs represents a useful starting point for biochemical analysis of lipid rafts. The physicochemical properties of DRMs present unique challenges to analysis of their protein composition. Existing methods of isolating DRM-enriched fractions involve flotation of cell extracts in a sucrose density gradient, which, although successful, can be labor intensive, time consuming and results in dilute sucrose-containing fractions with limited utility for direct proteomic analysis. In addition, several studies describing the proteomic characterization of DRMs using this and other approaches have reported the presence of nuclear proteins in such fractions. It is unclear whether these results reflect trafficking of nuclear proteins to DRMs or whether they arise from nuclear contamination during isolation. To address these issues, we have modified a published differential detergent extraction method to enable rapid DRM isolation that minimizes nuclear contamination and yields fractions compatible with mass spectrometry. DRM-enriched fractions isolated using the conventional or modified extraction methods displayed comparable profiles of known DRM-associated proteins, including flotillins, GPI-anchored proteins and heterotrimeric G-protein subunits. Thus, the modified procedure yielded fractions consistent with those isolated by existing methods. However, we observed a marked reduction in the percentage of nuclear proteins identified in DRM fractions isolated with the modified method (15%) compared to DRMs isolated by conventional means (36%). Furthermore, of the 21 nuclear proteins identified exclusively in modified DRM fractions, 16 have been reported to exist in other subcellular sites, with evidence to suggest shuttling of these species between the nucleus and other organelles. We describe a modified DRM isolation procedure that generates DRMs that are largely free of nuclear contamination and that is compatible with downstream proteomic analyses with minimal additional processing. Our findings also imply that identification of nuclear proteins in DRMs is likely to reflect legitimate movement of proteins between compartments, and is not a result of contamination during extraction.

Intrinsic fluorescence excitation-emission matrix spectral features of cottonseed protein fractions and the effects of denaturants

USDA-ARS?s Scientific Manuscript database

To better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation-emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, an...

Radioactive Lysine in Protein Metabolism Studies

DOE R&D Accomplishments Database

Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple,, G. H.

1950-01-09

Studies of incorporation of DL-lysine in various body proteins of the dog; the time course of labeled blood proteins; and apparent rate of disappearance of labeled plasma proteins for comparison of behavior of the plasma albumin and globulin fractions; shows more rapid turn over of globulin fraction.

Mapping Atheroprotective Functions and Related Proteins/Lipoproteins in Size Fractionated Human Plasma *

PubMed Central

Swertfeger, Debi K.; Li, Hailong; Rebholz, Sandra; Zhu, Xiaoting; Shah, Amy S.; Davidson, W. Sean; Lu, Long J.

2017-01-01

HDL has been shown to possess a variety of cardio-protective functions, including removal of excess cholesterol from the periphery, and inhibition of lipoprotein oxidation. It has been proposed that various HDL subparticles exist, each with distinct protein and lipid compositions, which may be responsible for HDL's many functions. We hypothesized that HDL functions will co-migrate with the operational lipoprotein subspecies when separated by gel filtration chromatography. Plasma from 10 healthy male donors was fractionated and the protein composition of the phospholipid containing fractions was analyzed by mass spectrometry (MS). Each fraction was evaluated for its proteomic content as well as its ability to promote cholesterol efflux and protect low density lipoprotein (LDL) from free radical oxidation. For each function, several peaks of activity were identified across the plasma size gradient. Neither cholesterol efflux or LDL antioxidation activity correlated strongly with any single protein across the fractions. However, we identified multiple proteins that had strong correlations (r values >0.7, p < 0.01) with individual peaks of activity. These proteins fell into diverse functional categories, including those traditionally associated with lipid metabolism, as well as alternative complement cascade, innate immunity and clotting cascades and immunoglobulins. Additionally, the phospholipid and cholesterol concentration of the fractions correlated strongly with cholesterol efflux (r = 0.95 and 0.82 respectively), whereas the total protein content of the fractions correlated best with antioxidant activity across all fractions (r = 0.746). Furthermore, two previously postulated subspecies (apoA-I, apoA-II and apoC-1; as well as apoA-I, apoC-I and apoJ) were found to have strong correlations with both cholesterol efflux and antioxidation activity. Up till now, very little has been known about how lipoprotein composition mediates functions like cholesterol efflux and antioxidation. PMID:28223350

Biochemistry of snake venom neurotoxins and their application to the study of synapse. [Neurotoxins isolated from venom of the Formosan banded krait

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanley, M.R.

1978-11-01

The crude venom of the Formosan banded krait, Bungarus multicinctus, was separated into eleven lethal protein fractions. Nine fractions were purified to final homogeneous toxins, designated ..cap alpha..-bungarotoxin, ..beta..-bungarotoxin, and toxins 7, 8, 9A, 11, 12, 13, and 14. Three of the toxins, ..cap alpha..-bungarotoxin, 7, and 8, were identified as post-synaptic curarimimetic neurotoxins. The remaining toxins were identified as pre-synaptic neurotoxins. ..cap alpha..-Bungarotoxin, toxin 7, and toxin 8 are all highly stable basic polypeptides of approx. 8000 daltons molecular weight. The pre-synaptic toxins fell into two structural groups: toxin 9A and 14 which were single basic chains of approx.more » 14,000 daltons, and ..beta..-bungarotoxin, and toxins 11 thru 13 which were composed of two chains of approx. 8000 and approx. 13,000 daltons covalently linked by disulfides. All the pre-synaptic neurotoxins were shown to have intrinsic calcium-dependent phospholipase A activities. Under certain conditions, intact synaptic membranes were hydrolyzed more rapidly than protein-free extracted synaptic-lipid liposomes which, in turn, were hydrolyzed more rapidly than any other tested liposomes. It was speculated that cell-surface arrays of phosphatidyl serine/glycolipids created high affinity target sites for ..beta..-bungarotoxin. Single-chain toxins were found to be qualitatively different from the two-chain toxins in their ability to block the functioning of acetylcholine receptors, and were quantitatively different in their enzymatic and membrane disruptive activities. ..beta..-Bungarotoxin was shown to be an extremely potent neuronal lesioning agent. There was no apparent selectivity for cholinergic over non-cholinergic neurons, nor for nerve terminals over cell bodies. It was suggested that ..beta..-bungarotoxin can be considered a useful new histological tool, which may exhibit some regional selectivity.« less

Food proteins and maturation of small intestinal microvillus membranes (MVM). II. Binding of gliadin hydrolysate fractions and of the gliadin peptide B3142.

PubMed

Stern, M; Gellermann, B; Belitz, H D; Wieser, H

1988-01-01

To investigate in vitro interactions between gliadin peptide fractions that have been shown to be toxic to celiac small intestinal mucosa in humans and small intestinal microvillus membranes (MVM) from rats during postnatal maturation, MVM were prepared from newborn, 18-day-old preweanling, and adult rats. Partially hydrolyzed gliadin peptide fractions B1-B4, and the pure gliadin peptide B3142 were radioiodinated and used for binding assays. Miniature ultracentrifugation was used for separation of unbound material. Binding of gliadin fractions to MVM was weak and nonspecific in terms of lacking saturation and inhibition. There was no inhibition of binding by mannan. Enzyme pretreatment of MVM (trypsin, neuraminidase, phospholipase C) did not result in any significant change of binding. Compared with peptides prepared from bovine serum albumin as a control, there was no significant difference in binding of gliadin peptide fractions to MVM. Thus, a lectin-like effect of gliadin peptides toward MVM, or the existence of a specific intestinal surface receptor for gliadin peptides appeared improbable. There were, however, consistent maturational changes in MVM binding in that newborn MVM bound more B1-B4 and B3142 compared with adult controls (p less than 0.001). Nonspecific binding of gliadin fractions to MVM might be related to the initiation of nonspecific in vitro effects of gliadin, particularly toward the immature small intestine. The MVM binding model in the rat clearly does not provide a system for studying celiac disease pathogenesis, but it might help clarify basic processes in the interaction between food-derived substances and elements of the gastrointestinal mucosal barrier.

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

PubMed

Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

2018-01-05

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.

A fast method for the determination of fractional contributions to solvation in proteins

PubMed Central

Talavera, David; Morreale, Antonio; Meyer, Tim; Hospital, Adam; Ferrer-Costa, Carles; Gelpi, Josep Lluis; de la Cruz, Xavier; Soliva, Robert; Luque, F. Javier; Orozco, Modesto

2006-01-01

A fast method for the calculation of residue contributions to protein solvation is presented. The approach uses the exposed polar and apolar surface of protein residues and has been parametrized from the fractional contributions to solvation determined from linear response theory coupled to molecular dynamics simulations. Application of the method to a large subset of proteins taken from the Protein Data Bank allowed us to compute the expected fractional solvation of residues. This information is used to discuss when a residue or a group of residues presents an uncommon solvation profile. PMID:17001031

Functional assessment of ubiquitin-depended processes under microgravity conditions

NASA Astrophysics Data System (ADS)

Zhabereva, Anastasia; Shenkman, Boris S.; Gainullin, Murat; Gurev, Eugeny; Kondratieva, Ekaterina; Kopylov, Arthur

Ubiquitylation, a widespread and important posttranslational modification of eukaryotic proteins, controls a multitude of critical cellular processes, both in normal and pathological conditions. The present work aims to study involvement of ubiquitin-dependent regulation in adaptive response to the external stimuli. Experiments were carried out on C57BL/6 mice. The microgravity state under conditions of real spaceflight on the biosatellite “BION-M1” was used as a model of stress impact. Additionally, number of control series including the vivarium control and experiments in Ground-based analog were also studied. The aggregate of endogenously ubiquitylated proteins was selected as specific feature of ubiquitin-dependent processes. Dynamic changes of modification pattern were characterized in liver tissue by combination of some methods, particularly by specific isolation of explicit protein pool, followed by immunodetection and/or mass spectrometry-based identification. The main approach includes specific extraction of proteins, modified by multiubiquitin chains of different length and topology. For this purpose two techniques were applied: 1) immunoprecipitation with antibodies against ubiquitin and/or multiubiquitin chains; 2) pull-down using synthetic protein construct termed Tandem Ubiquitin Binding Entities (TUBE, LifeSensors). TUBE represents fusion protein, composed of well characterized ubiquitin-binding domains, and thereby allows specific high-affinity binding and extraction of ubiquitylated proteins. Resulting protein fractions were analyzed by immunoblotting with antibodies against different types of multiubiquitin chains. Using this method we mapped endogenously modified proteins involved in two different types of ubiquitin-dependent processes, namely catabolic and non-catabolic ubiquitylation, in liver tissues, obtained from both control as well as experimental groups of animals, mentioned above. Then, isolated fractions of ubiquitylated proteins, were separated by SDS-PAGE and subjected for mass spectrometry-based analysis.With the described workflow, we identified more than 200 proteins including of 26S proteasome subunits, members of SUMO (Small Ubiquitin-like Modifier) family and ubiquitylated substrates. On the whole, our results provide an unbiased view of ubiquitylation state under microgravity conditions and thereby demonstrate the utility of proposed combination of analytical methods for functional assessment of ubiquitin-depended processes. Acknowledgment - We thank teams of Institute of Biomedical Problems of Russian Academy of Sciences and TsSKB “Progress” Samara for organization and preparation for spaceflight. This work is partially supported by the Russian Foundation for Basic Research (grant12-04-01836).

Expanding the bovine milk proteome through extensive fractionation.

PubMed

Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

2013-01-01

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

NASA Technical Reports Server (NTRS)

Mednieks, M. I.; Popova, I.; Grindeland, R. E.

1992-01-01

Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

Staircase and fractional part functions

NASA Astrophysics Data System (ADS)

Amram, Meirav; Dagan, Miriam; Ioshpe, Michael; Satianov, Pavel

2016-10-01

The staircase and fractional part functions are basic examples of real functions. They can be applied in several parts of mathematics, such as analysis, number theory, formulas for primes, and so on; in computer programming, the floor and ceiling functions are provided by a significant number of programming languages - they have some basic uses in various programming tasks. In this paper, we view the staircase and fractional part functions as a classical example of non-continuous real functions. We introduce some of their basic properties, present some interesting constructions concerning them, and explore some intriguing interpretations of such functions. Throughout the paper, we use these functions in order to explain basic concepts in a first calculus course, such as domain of definition, discontinuity, and oddness of functions. We also explain in detail how, after researching the properties of such functions, one can draw their graph; this is a crucial part in the process of understanding their nature. In the paper, we present some subjects that the first-year student in the exact sciences may not encounter. We try to clarify those subjects and show that such ideas are important in the understanding of non-continuous functions, as a part of studying analysis in general.

Supercritical carbon dioxide fractionation of whey protein isolate for new food-grade ingredients

USDA-ARS?s Scientific Manuscript database

A new, environmentally benign whey protein fractionation process was developed using supercritical CO2 (SCO2) as an acid aggregating agent to separate a-lactalbumin (a-LA) aggregates from soluble beta-lactoglobulin (beta-LG) protein in concentrated whey protein isolate (WPI) solutions. The process e...

Biochemical characterization of soluble proteins in pecan [Carya illinoinensis (Wangenh.) K. Koch].

PubMed

Venkatachalam, Mahesh; Roux, Kenneth H; Sathe, Shridhar K

2008-09-10

Pecans (cv. Desirable) contained approximately 10% protein on a dry weight basis. The minimum nitrogen solubility (5.9-7.5%) at 0.25-0.75 M trichloroacetic acid represented the nonprotein nitrogen. Among the solvents assessed for protein solubilization, 0.1 M NaOH was the most effective, while borate saline buffer (pH 8.45) was judged to be optimal for protein solubilization. The protein solubility was minimal in the pH range of 3-7 and significantly increased on either side of this pH range. Increasing the NaCl concentration from 0 to 4 M significantly improved ( approximately 8-fold increase) protein solubilization. Following Osborne protein fractionation, the alkali-soluble glutelin fraction (60.1%) accounted for a major portion of pecan proteins followed by globulin (31.5%), prolamin (3.4%), and albumin (1.5%), respectively. The majority of pecan polypeptides were in the molecular mass range of 12-66 kDa and in the pI range of 4.0-8.3. The pecan globulin fraction was characterized by the presence of several glycoprotein polypeptides. Lysine was the first limiting essential amino acid in the defatted flour, globulin, prolamin, and alkaline glutelin fractions. Leucine and tryptophan were the first limiting essential amino acids in albumin and acid glutelin fractions, respectively. Rabbit polyclonal antibodies detected a range of pecan polypeptides in the 12-60 kDa range, of which the globulin fraction contained the most reactive polypeptides.

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

PubMed

Wang, Zhen; Schey, Kevin L

2015-12-01

Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

Fundamental considerations in the effect of molecular weight on the glass transition of the gelatin/cosolute system.

PubMed

Jiang, Bin; Kasapis, Stefan; Kontogiorgos, Vassilis

2012-05-01

Four molecular fractions of gelatin produced by alkaline hydrolysis of collagen were investigated in the presence of cosolute to record the mechanical properties of the glass transition in high-solid preparations. Dynamic oscillatory and stress relaxation moduli in shear were recorded from 40°C to temperatures as low as -60°C. The small-deformation behavior of these linear polymers was separated by the method of reduced variables into a basic function of time alone and a basic function of temperature alone. The former allowed the reduction of isothermal runs into a master curve covering 17 orders of magnitude in the time domain. The latter follows the passage from the rubbery plateau through the glass transition region to the glassy state seen in the variation of shift factor, a(T) , as a function of temperature. The mechanical glass transition temperature (T(g) ) is pinpointed at the operational threshold of the free volume theory and the predictions of the reaction rate theory. Additional insights into molecular dynamics are obtained via the coupling model of cooperativity, which introduces the concept of coupling constant or interaction strength of local segmental motions that govern structural relaxation at the vicinity of T(g) . The molecular weight of the four gelatin fractions appears to have a profound effect on the transition temperature or coupling constant of vitrified matrices, as does the protein chemistry in relation to that of amorphous synthetic polymers or gelling polysaccharides. © 2011 Wiley Periodicals, Inc.

Composition and molecular weight distribution of carob germ protein fractions.

PubMed

Smith, Brennan M; Bean, Scott R; Schober, Tilman J; Tilley, Michael; Herald, Thomas J; Aramouni, Fadi

2010-07-14

Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) coupled with multiangle laser light scattering (SEC-MALS), and electrophoretic analysis. Using a modified Osborne extraction procedure, carob germ flour proteins were found to contain approximately 32% albumin and globulin and approximately 68% glutelin with no prolamins detected. The albumin and globulin fraction was found to contain low amounts of disulfide-bonded polymers with relatively low M(w) ranging up to 5 x 10(6) Da. The glutelin fraction, however, was found to contain large amounts of high molecular weight disulfide-bonded polymers with M(w) up to 8 x 10(7) Da. When extracted under nonreducing conditions and divided into soluble and insoluble proteins as typically done for wheat gluten, carob germ proteins were found to be almost entirely ( approximately 95%) in the soluble fraction with only ( approximately 5%) in the insoluble fraction. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater M(w) than the soluble proteins and ranged up to 8 x 10(7) Da. The lower M(w) distribution of the polymeric proteins of carob germ flour may account for differences in functionality between wheat and carob germ flour.

Effects of N-acetyl-L-cysteine on redox status and markers of renal function in mice inoculated with Bothrops jararaca and Crotalus durissus terrificus venoms.

PubMed

Barone, Juliana Marton; Frezzatti, Rodrigo; Silveira, Paulo Flavio

2014-03-01

Renal dysfunction is an important aggravating factor in accidents caused by Crotalus durissus terrificus (Cdt) and Bothrops jararaca (Bj) bites. N-acetyl-l-cysteine (NAC) is well known as a nephroprotective antioxidant with low toxicity. The present study investigated the effects of NAC on redox status and markers of renal function in mice that received vehicle (controls) or venoms (v) of Cdt and Bj. In controls NAC promoted hypercreatinemia, hypouremia, hyperosmolality with decreased urea in urine, hyperproteinuria, decreased protein and increased dipeptidyl peptidase IV (DPPIV) in membrane-bound fraction (MF) from renal cortex (RC) and medulla (RM). NAC ameliorated or normalized altered creatinuria, proteinemia and aminopeptidase (AP) acid in MF, AP basic (APB) in soluble fraction (SF), and neutral AP in SF and MF from RC and RM in vBj envenomation. NAC ameliorated or normalized altered neutral AP in SF from RC and RM, and DPPIV and protein in MF from RC in vCdt envenomation. NAC ameliorated or restored renal redox status respectively in vCdt and vBj, and normalized uricemia in both envenomations. These data are promising perspectives that recommend the clinical evaluation of NAC as potential coadjuvant in the anti venom serotherapy for accidents with these snake's genera. Copyright © 2014 Elsevier Ltd. All rights reserved.

Oligodendrocyte progenitor cells proliferate and survive in an immature state following treatment with an axolemma-enriched fraction

PubMed Central

Becker-Catania, Sara G; Nelson, Julie K; Olivares, Shantel; Chen, Shu-Jen; DeVries, George H

2011-01-01

The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation. PMID:21345173

The impact of aging wine in high and low oxygen conditions on the fractionation of Cu and Fe in Chardonnay wine.

PubMed

Kontoudakis, Nikolaos; Guo, Anque; Scollary, Geoffrey R; Clark, Andrew C

2017-08-15

Solid-phase extraction has previously been used to fractionate copper and iron into hydrophobic, cationic and residual forms. This study showed the change in fractionated copper and iron in Chardonnay wines with 1-year of bottle aging under variable oxygen and protein concentrations. Wines containing protein in low oxygen conditions induced a decrease (20-50%) in total copper and increased the proportion of the hydrophobic copper fraction, associated with copper(I) sulfide. In contrast, protein stabilised wines showed a lower proportion of the hydrophobic copper fraction after 1-year of aging. In oxidative storage conditions, the total iron decreased by 60% when at high concentration, and the concentration of the residual fraction of both copper and iron increased. The results show that oxidative storage increases the most oxidative catalytic form of the metal, whilst changes during reductive storage depend on the extent of protein stabilisation of the wine. Copyright © 2017 Elsevier Ltd. All rights reserved.

Se metallomics during lactic fermentation of Se-enriched yogurt.

PubMed

Palomo, María; Gutiérrez, Ana M; Pérez-Conde, M Concepción; Cámara, Carmen; Madrid, Yolanda

2014-12-01

Selenium biotransformation by lactic acid bacteria during the preparation of Se-enriched yogurt was evaluated. The study focused on the distribution of selenium in the aqueous soluble protein fraction and the detection of selenoamino acids. Screening of selenium in Tris-buffer-urea soluble fraction was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis after pre-fractionating with asymmetric field flow fractionation using inductively coupled plasma-mass spectrometry as the detector. Selenium-containing fractions were identified by peptide mapping using nano LC-ESI/LTQMS. Proteins such as thioredoxin, glutaredoxin, albumin, β-lactoglobulin, and lactoperoxidase were identified in the selenium-containing fraction. All these proteins were detected in both the control and the selenium-enriched yogurt except chaperones, which were only detected in the control samples. Chaperones are heat-shock proteins expressed in response to elevated temperature or other cellular stresses. Selenium may have an effect on chaperones expression in Lactobacillus. For the amino acids analysis, selenocysteine was the primary seleno-containing species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Qualitative and Quantitative Analysis of Proteome and Peptidome of Human Follicular Fluid Using Multiple Samples from Single Donor with LC-MS and SWATH Methodology.

PubMed

Lewandowska, Aleksandra E; Macur, Katarzyna; Czaplewska, Paulina; Liss, Joanna; Łukaszuk, Krzysztof; Ołdziej, Stanisław

2017-08-04

Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small-scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient-specific. Ultrafiltration was used to fractionate hFF to high-molecular-weight (HMW) proteome (>10 kDa) and low-molecular-weight (LMW) peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, from which 59 were never reported before as hFF components. 55 (45 not reported before) proteins were found by analyzing LMW fraction, 67 (14 not reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 11 proteins varied substantially among hFF samples from single donors, and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.

PLASMA PROTEIN ELECTROPHORESIS AND SELECT ACUTE PHASE PROTEINS IN HEALTHY BONNETHEAD SHARKS (SPHYRNA TIBURO) UNDER MANAGED CARE.

PubMed

Hyatt, Michael W; Field, Cara L; Clauss, Tonya M; Arheart, Kristopher L; Cray, Carolyn

2016-12-01

Preventative health care of elasmobranchs is an important but understudied field of aquatic veterinary medicine. Evaluation of inflammation through the acute phase response is a valuable tool in health assessments. To better assess the health of bonnethead sharks ( Sphyrna tiburo ) under managed care, normal reference intervals of protein electrophoresis (EPH) and the acute phase proteins, C-reactive protein (CRP) and haptoglobin (HP), were established. Blood was collected from wild caught, captive raised bonnethead sharks housed at public aquaria. Lithium heparinized plasma was either submitted fresh or stored at -80°C prior to submission. Electrophoresis identified protein fractions with migration characteristics similar to other animals with albumin, α-1 globulin, α-2 globulin, β globulin, and γ globulin. These fractions were classified as fractions 1-5 as fractional contents are unknown in this species. Commercial reagents for CRP and HP were validated for use in bonnethead sharks. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology for the calculation of 90% reference intervals. Once established, the diagnostic and clinical applicability of these reference intervals was used to assess blood from individuals with known infectious diseases that resulted in systemic inflammation and eventual death. Unhealthy bonnethead sharks had significantly decreased fraction 2, fraction 3, and fraction 3:4 ratio and significantly increased fraction 5, CRP, and HP. These findings advance our understanding of elasmobranch acute phase inflammatory response and health and aid clinicians in the diagnosis of inflammatory disease in bonnethead sharks.

Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9.

PubMed

Köhler, Thomas; Weber, Ingrid; Glaubitz, Clemens; Wachtveitl, Josef

2017-05-01

The retinal protein proteorhodopsin is a homolog of the well-characterized light-driven proton pump bacteriorhodopsin. Basic mechanisms of proton transport seem to be conserved, but there are noticeable differences in the pH ranges of proton transport. Proton transport and protonation state of a carboxylic acid side chain, the primary proton acceptor, are correlated. In case of proteorhodopsin, the pK a of the primary proton acceptor Asp-97 (pK a  ≈ 7.5) is unexpectedly close to environmental pH (pH ≈ 8). A significant fraction of proteorhodopsin is possibly inactive at natural pH, in contrast to bacteriorhodopsin. We investigated photoinduced kinetics of proteorhodopsin between pH 5 and pH 9 by time resolved UV/vis absorption spectroscopy. Kinetics is inhomogeneous within that pH region and can be considered as a superposition of two fractions. These fractions are correlated with the Asp-97 titration curve. Beside Asp-97, protonation equilibria of other groups influence kinetics, but the observations do not point toward major differences of primary proton acceptor function in proteorhodopsin and bacteriorhodopsin. The pK a of proteorhodopsin and some of its variants is suspected to be an example of molecular adaptation to the physiology of the original organisms. © 2017 The American Society of Photobiology.

Periplasmic Proteins of the Extremophile Acidithiobacillus ferrooxidans

PubMed Central

Chi, An; Valenzuela, Lissette; Beard, Simon; Mackey, Aaron J.; Shabanowitz, Jeffrey; Hunt, Donald F.; Jerez, Carlos A.

2015-01-01

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of obtaining energy by oxidizing ferrous iron or sulfur compounds such as metal sulfides. Some of the proteins involved in these oxidations have been described as forming part of the periplasm of this extremophile. The detailed study of the periplasmic components constitutes an important area to understand the physiology and environmental interactions of microorganisms. Proteomics analysis of the periplasmic fraction of A. ferrooxidans ATCC 23270 was performed by using high resolution linear ion trap-FT MS. We identified a total of 131 proteins in the periplasm of the microorganism grown in thiosulfate. When possible, functional categories were assigned to the proteins: 13.8% were transport and binding proteins, 14.6% were several kinds of cell envelope proteins, 10.8% were involved in energy metabolism, 10% were related to protein fate and folding, 10% were proteins with unknown functions, and 26.1% were proteins without homologues in databases. These last proteins are most likely characteristic of A. ferrooxidans and may have important roles yet to be assigned. The majority of the periplasmic proteins from A. ferrooxidans were very basic compared with those of neutrophilic microorganisms such as Escherichia coli, suggesting a special adaptation of the chemolithoautotrophic bacterium to its very acidic environment. The high throughput proteomics approach used here not only helps to understand the physiology of this extreme acidophile but also offers an important contribution to the functional annotation for the available genomes of biomining microorganisms such as A. ferrooxidans for which no efficient genetic systems are available to disrupt genes by procedures such as homologous recombination. PMID:17911085

CELLULAR AND SECRETORY PROTEINS OF THE SALIVARY GLANDS OF SCIARA COPROPHILA DURING THE LARVAL-PUPAL TRANSFORMATION

PubMed Central

Been, Anita C.; Rasch, Ellen M.

1972-01-01

The cellular and secretory proteins of the salivary gland of Sciara coprophila during the stages of the larval-pupal transformation were examined by electrophoresis in 0.6 mm sheets of polyacrylamide gel with both SDS-continuous and discontinuous buffer systems. After SDS-electrophoresis, all electrophoretograms of both reduced and nonreduced proteins from single glands stained with Coomassie brilliant blue revealed a pattern containing the same 25 bands during the stages of the larval-pupal transformation. With the staining procedures used in this study, qualitative increases and decreases were detected in existing proteins and enzymes. There was no evidence, however, for the appearance of new protein species that could be correlated with the onset of either pupation or gland histolysis. Electrophoretograms of reduced samples of anterior versus posterior gland parts indicated that no protein in the basic pattern of 25 bands was unique to either the anterior or posterior gland part. Electrophoretograms of reduced samples of secretion collected from either actively feeding or "cocoon"-building animals showed an electrophoretic pattern containing up to six of the 25 protein fractions detected in salivary gland samples, with varied amounts of these same six proteins in electrophoretograms of secretion samples from a given stage. Zymograms of non-specific esterases in salivary gland samples revealed a progressive increase in the amount of esterase reaction produce in one major band and some decrease in the second major band during later stages of the larval-pupal transformation. PMID:4116523

Effect of nitrogen nutrition on endosperm protein synthesis in wild and cultivated barley grown in spike culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corke, H.; Atsmon, D.

1988-06-01

In normal growth conditions, total protein percent, in the endosperm at maturity in barley cultivar Hordeum vulgare L. cv Ruth was about 14%, whereas in an accession of wild barley, Hordeum spontaneum Koch line 297, it was about 28%. Spike culture experiments were conducted to ascertain whether there were basic differences between the two genotypes under conditions of widely different nitrogen supply. Spikes of each genotype were grown from 8 to 25 days after flowering in in vitro culture in a growth medium containing 0 to 4 grams per liter nitrogen supplied as NH{sub 4}NO{sub 3}. Spikes were pulse-labeled atmore » intervals from 12 to 24 days after flowering with 3.7 megabecquerel of ({sup 3}H)leucine to determine relative rates of synthesis of hordein-1 and hordein-2 polypedtides. At low nitrogen levels Ruth had a lower protein content than 297, but at increasing nitrogen levels its protein content increased rapidly and reached a maximum (35%) higher than 297 (30%). The relative contribution of the hordein fraction to total protein increased mainly with time, and hordein-1 to total hordein increased mainly with nitrogen level, in both genotypes. There appeared to be no fundamental limitations in the capacity of Ruth to accumulate protein: 297 appears to have a greater basal level of nitrogen availability under normal conditions.« less

The effect of dietary protein and lipid source on dorsal fin erosion in rainbow trout, Oncorhynchus mykiss

USGS Publications Warehouse

Barrows, F.T.; Lellis, W.A.

1999-01-01

A study was conducted to determine the effect of dietary protein and lipid source on dorsal fin erosion in rainbow trout. Seven diets were each fed to four replicate lots of 300 first-feeding fry cultured in 75 1 aluminum troughs for 8 weeks. Two basal diets were manufactured with approximately equal nutrient content, one using krill and squid meals and the other anchovy meal as the primary protein-containing ingredients. The meals used to manufacture the diets were separated into two fractions: lipid (ether-extractable); and protein/ash (non-ether-extractable) using a large soxhlet. The fractions were then recombined to create two additional diets; one containing anchovy protein/ash with krill/squid lipid, the other krill/squid protein/ash with fish lipid. A fifth diet recombined krill/squid protein/ash with krill/squid lipid to evaluate effects of the extraction process. Two additional treatments included a diet with a portion of the krill meal replaced by poultry by-product meal, and the basal anchovy meal diet supplemented with sodium, magnesium, and copper. Fish consuming diets containing anchovy meal as the primary protein source gained more weight (P < 0.05) than fish consuming krill/squid meal-based diets. Dorsal fin index (DFI, measured as mean dorsal fin height x 100/total fish length) was greater (P < 0.05) for fish consuming diets containing krill/squid meal protein/ash fraction (DFI = 9.9%-10.0%) than for fish consuming diets containing anchovy meal protein/ash fraction (DFI = 4.9%-5.3%), regardless of lipid source. Supplementation of the anchovy meal diet with sodium, magnesium, and copper improved (P < 0.05) DFI by approximately 20%, but not to the level supported by the krill/squid meal protein/ash fraction diets. The cost of the krill meal diet was reduced by inclusion of poultry by-product meal without affecting dorsal fin condition. These data indicate that the dietary agent contributing to dorsal fin erosion in rainbow trout is not present in the ether-extractable fraction of the diet, but rather in the protein or mineral fraction.

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 2: Computer controlled pH gradients in the presence of urea provide improved separation of proteins: Stability influenced anion and cation exchange chromatography.

PubMed

Hirsh, Allen G; Tsonev, Latchezar I

2017-04-28

This paper details the use of a method of creating controlled pH gradients (pISep) to improve the separation of protein isoforms on ion exchange (IEX) stationary phases in the presence of various isocratic levels of urea. The pISep technology enables the development of computer controlled pH gradients on both cationic (CEX) and anionic (AEX) IEX stationary phases over the very wide pH range from 2 to 12. In pISep, titration curves generated by proportional mixing of the acidic and basic pISep working buffers alone, or in the presence of non-buffering solutes such as the neutral salt NaCl (0-1M), polar organics such as urea (0-8M) or acetonitrile (0-80 Vol%), can be fitted with high fidelity using high order polynomials which, in turn allows construction of a mathematical manifold %A (% acidic pISep buffer) vs. pH vs. [non-buffering solute], permitting precise computer control of pH and the non-buffering solute concentration allowing formation of dual uncoupled liquid chromatographic (LC) gradients of arbitrary shape (Hirsh and Tsonev, 2012 [1]). The separation of protein isoforms examined in this paper by use of such pH gradients in the presence of urea demonstrates the fractionation power of a true single step two dimensional liquid chromatography which we denote as Stability-Influenced Ion Exchange Chromatography (SIIEX). We present evidence that SIIEX is capable of increasing the resolution of protein isoforms difficult to separate by ordinary pH gradient IEX, and potentially simplifying the development of laboratory and production purification strategies involving on-column simultaneous pH and urea unfolding or refolding of targeted proteins. We model some of the physics implied by the dynamics of the observed protein fractionations as a function of both urea concentration and pH assuming that urea-induced native state unfolding competes with native state electrostatic interaction binding to an IEX stationary phase. Implications for in vivo protein-membrane interactions are discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

NASA Technical Reports Server (NTRS)

Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

1980-01-01

Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

Academic Music: Music Instruction to Engage Third-Grade Students in Learning Basic Fraction Concepts

ERIC Educational Resources Information Center

Courey, Susan Joan; Balogh, Endre; Siker, Jody Rebecca; Paik, Jae

2012-01-01

This study examined the effects of an academic music intervention on conceptual understanding of music notation, fraction symbols, fraction size, and equivalency of third graders from a multicultural, mixed socio-economic public school setting. Students (N = 67) were assigned by class to their general education mathematics program or to receive…

Allergenicity, trypsin inhibitor activity and nutritive quality of enzymatically modified soy proteins.

PubMed

De La Barca, Ana María Calderón; Wall, Abraham; López-Díaz, José Alberto

2005-05-01

Two ultrafiltered soy flour protein fractions were evaluated; the first was obtained by hydrolysis (0.5-3 kDa, F(0.5-3)), and the second was an enzymatically methionine-enriched fraction (1-10 kDa, F(1-10)E). Amino acid profiles, protein quality, allergenicity (against soy-sensitive infant sera) and trypsin inhibitor activity were determined. Fraction F(1-10)E fulfilled amino acid requirements for infants, whereas the F(0.5-3) fraction was methionine deficient. Both fractions were similar in net protein utilization, and F(1-10)E digestibility was comparable with casein and higher (P?

THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS

PubMed Central

A, Güllü; S, Dede

2016-01-01

Background: TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Materials and methods: Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. Results: The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control’s (p<0.05). In addition, it was observed that the γ-globulin levels of TQ group were lower than that of the control group’s (p<0.05). Conclusion: From the results, it was observed that the changes of these fractions may have originated from elevation or decline synthesis, or activities of containing proteins. PMID:28480357

Fractionation, partial characterization and bioactivity of water-soluble polysaccharides and polysaccharide-protein complexes from Pleurotus geesteranus.

PubMed

Zhang, Mei; Zhu, Lin; Cui, Steve W; Wang, Qi; Zhou, Ting; Shen, Hengsheng

2011-01-01

Fractionation and purification of mushroom polysaccharides is a critical process for mushroom clinical application. After a hot-water treatment, the crude Pleurotus geesteranus (PG) was further fractionated into four fractions (PG-1, -2, -3, -4) using gradient precipitation with water and ammonia sulphate. By controlling the initial polymer concentration and ratio of solvents, this process produced PG fractions with high chemical uniformity and narrow Mw distribution without free proteins. Structurally, PG-1 and PG-2 are pure homopolysaccharide mainly composed of glucose; and PG-3 and PG-4 are heteropolysaccharide-protein complexes. PG-2, a high M(w) fraction mainly composed of glucose presented significant cytotoxicity at the concentration of 200 and 100 μg/ml to human breast cancer cells. Here, we report a new mushroom polysaccharides extraction and fractionation method, with which we produced four fractions of PG with PG-2 appearing effective anti-tumour activity. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romaen, R.

A protein fraction capable of inhibit in vitro protein synthesis was found in potato tubers in fresh and wounded tissue. Inhibitor activity from fresh tissue decays with wounding. Inhibition activity was detected absorbed to ribsomal fraction and cytosol of potato tuber tissue by a partially reconstituted in vitro system from potato tuber and wheat germ. Adsorbed ribosomal fraction was more suitable of purification. This fraction was washed from ribosomes with 0.3M KCl, concentrated with ammonium sulfate precipitation and purified through sephadex G100 and sephadex G-75 columns chromatography. After 61 fold purification adsorbed protein fraction can inhibit germination of maize, wheatmore » and sesame seeds, as well as {sup 3}H-leucine incorporation into protein by imbibed maize embryos. Inhibition activity was lost by temperature, alkali and protease-K hydrolysis. Preliminar analysis could not show presence of reductor sugars. Physiological role of this inhibitor in relation to rest and active tissue remains to be studied.« less

Fractionation of distillers dried grains with solubles (DDGS) by sieving and winnowing.

PubMed

Liu, KeShun

2009-12-01

Four commercial samples of distillers dried grains with solubles (DDGS) were sieved. All sieved fractions except for the pan fraction, constituting about 90% of original mass, were then winnowed with an air blast seed cleaner. Sieving was effective in producing fractions with varying composition. As the particle size decreased, protein and ash contents increased, and total carbohydrate (CHO) decreased. Winnowing sieved fractions was also effective in shifting composition, particularly for larger particle classes. Heavy sub-fractions were enriched in protein, oil and ash, while light sub-fractions were enriched for CHO. For protein, the combination of the two procedures resulted in a maximum 56.4% reduction in a fraction and maximum 60.2% increase in another fraction. As airflow velocity increased, light sub-fraction mass increased, while the compositional difference between the heavy and light sub-fractions decreased. Winnowing three times at a lower velocity was as effective as winnowing one time at a medium velocity. Winnowing the whole DDGS was much less effective than winnowing sieved fractions in changing composition, but sieving winnowed fractions was more effective than sieving whole DDGS. The two combination sequences gave comparable overall effects but sieving followed by winnowing is recommended because it requires less time. Regardless of combinational sequence, the second procedure was more effective in shifting composition than the first procedure.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue.

PubMed

Pennington, Kyla; McGregor, Emma; Beasley, Clare L; Everall, Ian; Cotter, David; Dunn, Michael J

2004-01-01

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.

Two Bioactive Molecular Weight Fractions of a Conditioned Medium Enhance RPE Cell Survival on Age-Related Macular Degeneration and Aged Bruch's Membrane

PubMed Central

Sugino, Ilene K.; Sun, Qian; Springer, Carola; Cheewatrakoolpong, Noounanong; Liu, Tong; Li, Hong; Zarbin, Marco A.

2016-01-01

Purpose To characterize molecular weight fractions of bovine corneal endothelial cell conditioned medium (CM) supporting retinal pigment epithelium (RPE) cell survival on aged and age-related macular degeneration (AMD) Bruch's membrane. Methods CM was subject to size separation using centrifugal filters. Retentate and filtrate fractions were tested for bioactivity by analyzing RPE survival on submacular Bruch's membrane of aged and AMD donor eyes and behavior on collagen I-coated tissue culture wells. Protein and peptide composition of active fractions was determined by mass spectrometry. Results Two bioactive fractions, 3-kDa filtrate and a 10-50–kDa fraction, were necessary for RPE survival on aged and AMD Bruch's membrane. The 3-kDa filtrate, but not the 10-50–kDa fraction, supported RPE growth on collagen 1‐coated tissue culture plates. Mass spectrometry of the 10-50–kDa fraction identified 175 extracellular proteins, including growth factors and extracellular matrix molecules. Transforming growth factor (TGF)β-2 was identified as unique to active CM. Peptides representing 29 unique proteins were identified in the 3-KDa filtrate. Conclusions These results indicate there is a minimum of two bioactive molecules in CM, one found in the 3-kDa filtrate and one in the 10-50–kDa fraction, and that bioactive molecules in both fractions must be present to ensure RPE survival on Bruch's membrane. Mass spectrometry analysis suggested proteins to test in future studies to identify proteins that may contribute to CM bioactivity. Translational Relevance Results of this study are the first steps in development of an adjunct to cell-based therapy to ensure cell transplant survival and functionality in AMD patients. PMID:26933521

Fractionation of oats into products enriched with protein, beta-glucan, starch, or other carbohydrates

USDA-ARS?s Scientific Manuscript database

A modified wet method was developed to fractionate ground oat groats into 4 fractions enriched with beta-glucan (BG), protein, starch, and other carbohydrates (CHO), respectively. Effects of defatting oats and centrifuge force for separation were also investigated. Results show that, depending on ...

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gritsenko, Marina A.; Xu, Zhe; Liu, Tao

Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

2016-01-01

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

[L-arginine metabolism enzyme activities in rat liver subcellular fractions under condition of protein deprivation].

PubMed

Kopyl'chuk, G P; Buchkovskaia, I M

2014-01-01

The features of arginase and NO-synthase pathways of arginine's metabolism have been studied in rat liver subcellular fractions under condition of protein deprivation. During the experimental period (28 days) albino male rats were kept on semi synthetic casein diet AIN-93. The protein deprivation conditions were designed as total absence of protein in the diet and consumption of the diet partially deprived with 1/2 of the casein amount compared to in the regular diet. Daily diet consumption was regulated according to the pair feeding approach. It has been shown that the changes of enzyme activities, involved in L-arginine metabolism, were characterized by 1.4-1.7 fold decrease in arginase activity, accompanied with unchanged NO-synthase activity in cytosol. In mitochondrial fraction the unchanged arginase activity was accompanied by 3-5 fold increase of NO-synthase activity. At the terminal stages of the experiment the monodirectional dynamics in the studied activities have been observed in the mitochondrial and cytosolfractions in both experimental groups. In the studied subcellular fractions arginase activity decreased (2.4-2.7 fold with no protein in the diet and 1.5 fold with partly supplied protein) and was accompanied by NO-synthase activity increase by 3.8 fold in cytosole fraction, by 7.2 fold in mitochondrial fraction in the group with no protein in the diet and by 2.2 and 3.5 fold in the group partialy supplied with protein respectively. The observed tendency is presumably caused by the switch of L-arginine metabolism from arginase into oxidizing NO-synthase parthway.

Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, S.; Cole, R.; Chiappelli, F.

During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase inmore » mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.« less

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

NASA Astrophysics Data System (ADS)

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 and 2nd amide groups (P < 0.10), but no difference in the 1st to 2nd amide group intensity ratios (P > 0.05). These results indicate that the sourced-origins and the internal molecular structure profiles affected biological functions, nutrient bioavailability and biodegradation.

The rat red blood cell proteome is altered by priming with 2-butoxyethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palkar, Prajakta S.; Kakhniashvili, David G.; Goodman, Steven R.

2008-08-01

Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD{sub 90} dose (1500 mg BE/kg, p.o.) offers protection against the lethal dose-induced hemolysis and death in female Sprague Dawley rats because of prompt and efficient replacement of red blood cells (RBCs) with new resilient RBCs. The objective of the present work was to analyze the altered proteome of RBCs upon priming with BE in order to identify the potential anti-hemolytic survival proteins induced in the primed rat RBCs (P-RBCs) as opposed to vehicle-treated RBCs (V-RBCs). The RBCs from the two groupsmore » were fractionated into membrane and cytosolic fractions. The cytosolic fractions were further fractionated for proteomic analysis into 3 fractions. The fractions were labeled with Cy3 and Cy5 fluorescent dyes and subjected to 2-dimensional differential gel electrophoresis (DIGE) to analyze the protein profiles. Seven membrane and 8 cytosolic proteins were found to be significantly increased ({>=} 2.5 fold) in P-RBCs as compared to V-RBCs. The identified proteins can be classified into antioxidant, membrane skeleton, protein turnover, lipid raft, and energy metabolism components. Increased levels of the proteins from antioxidant and membrane skeleton groups were confirmed by Western blot analysis. The study provides the first report on protein profiling of rat RBCs as well as on alteration of the proteome upon exposure to a priming dose of hemotoxicant. Further studies are needed to prove the protective role of the identified proteins and will initiate the field of survival/protective/anti-hemolytic proteins in RBCs.« less

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells

PubMed Central

Wang, Zhen; Schey, Kevin L.

2015-01-01

Purpose Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids—key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Methods Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. Results A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. Conclusions These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells. PMID:26747763

Protein characterization of protein bodies from cotyledons of Mucuna pruriens (L.) DC.

PubMed

Bellani, Lorenza; Giglioni, Stefania; Muccifora, Simonetta

2013-03-01

Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5-12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PubMed Central

Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

Role of foam drainage in producing protein aggregates in foam fractionation.

PubMed

Li, Rui; Zhang, Yuran; Chang, Yunkang; Wu, Zhaoliang; Wang, Yanji; Chen, Xiang'e; Wang, Tao

2017-10-01

It is essential to obtain a clear understanding of the foam-induced protein aggregation to reduce the loss of protein functionality in foam fractionation. The major effort of this work is to explore the roles of foam drainage in protein aggregation in the entire process of foam fractionation with bovine serum albumin (BSA) as a model protein. The results show that enhancing foam drainage increased the desorption of BSA molecules from the gas-liquid interface and the local concentration of desorbed molecules in foam. Therefore, it intensified the aggregation of BSA in foam fractionation. Simultaneously, it also accelerated the flow of BSA aggregates from rising foam into the residual solution along with the drained liquid. Because enhancing foam drainage increased the relative content of BSA molecules adsorbed at the gas-liquid interface, it also intensified the aggregation of BSA during both the defoaming process and the storage of the foamate. Furthermore, enhancing foam drainage more readily resulted in the formation of insoluble BSA aggregates. The results are highly important for a better understanding of foam-induced protein aggregation in foam fractionation. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.

PubMed

Park, Seong-Jun; Ahn, Hee-Sung; Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.

Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

PubMed

Marini, Juan C; Didelija, Inka Cajo

2015-01-01

Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

The Nature of the Dietary Protein Impacts the Tissue-to-Diet 15N Discrimination Factors in Laboratory Rats

PubMed Central

Poupin, Nathalie; Bos, Cécile; Mariotti, François; Huneau, Jean-François; Tomé, Daniel; Fouillet, Hélène

2011-01-01

Due to the existence of isotope effects on some metabolic pathways of amino acid and protein metabolism, animal tissues are 15N-enriched relative to their dietary nitrogen sources and this 15N enrichment varies among different tissues and metabolic pools. The magnitude of the tissue-to-diet discrimination (Δ15N) has also been shown to depend on dietary factors. Since dietary protein sources affect amino acid and protein metabolism, we hypothesized that they would impact this discrimination factor, with selective effects at the tissue level. To test this hypothesis, we investigated in rats the influence of a milk or soy protein-based diet on Δ15N in various nitrogen fractions (urea, protein and non-protein fractions) of blood and tissues, focusing on visceral tissues. Regardless of the diet, the different protein fractions of blood and tissues were generally 15N-enriched relative to their non-protein fraction and to the diet (Δ15N>0), with large variations in the Δ15N between tissue proteins. Δ15N values were markedly lower in tissue proteins of rats fed milk proteins compared to those fed soy proteins, in all sampled tissues except in the intestine, and the amplitude of Δ15N differences between diets differed between tissues. Both between-tissue and between-diet Δ15N differences are probably related to modulations of the relative orientation of dietary and endogenous amino acids in the different metabolic pathways. More specifically, the smaller Δ15N values observed in tissue proteins with milk than soy dietary protein may be due to a slightly more direct channeling of dietary amino acids for tissue protein renewal and to a lower recycling of amino acids through fractionating pathways. In conclusion, the present data indicate that natural Δ15N of tissue are sensitive markers of the specific subtle regional modifications of the protein and amino acid metabolism induced by the protein dietary source. PMID:22132207

Three-dimensional fluorescence analysis of chernozem humic acids and their electrophoretic fractions

NASA Astrophysics Data System (ADS)

Trubetskoi, O. A.; Trubetskaya, O. E.

2017-09-01

Polyacrylamide gel electrophoresis in combination with size-exclusion chromatography (SEC-PAGE) has been used to obtain stable electrophoretic fractions of different molecular size (MS) from chernozem humic acids (HAs). Three-dimensional fluorescence charts of chernozem HAs and their fractions have been obtained for the first time, and all fluorescence excitation-emission maxima have been identified in the excitation wavelength range of 250-500 nm. It has been found that fractionation by the SEC-PAGE method results in a nonuniform distribution of protein- and humin-like fluorescence of the original HA preparation among the electrophoretic fractions. The electrophoretic fractions of the highest and medium MSs have only the main protein-like fluorescence maximum and traces of humin-like fluorescence. In the electrophoretic fraction of the lowest MS, the intensity of protein-like fluorescence is low, but the major part of humin-like fluorescence is localized there. Relationships between the intensity of protein-like fluorescence and the weight distribution of amino acids have been revealed, as well as between the degree of aromaticity and the intensity of humin-like fluorescence in electrophoretic fractions of different MSs. The obtained relationships can be useful in the interpretation of the spatial structural organization and ecological functions of soil HAs.

Purification and characterization of an antioxidant glycoprotein from the hydrolysate of Mustelus griseus.

PubMed

Li, Zhongrui; Wang, Bin; Chi, Changfeng; Gong, Yandan; Tang, Jiajia; Luo, Hongyu

2013-01-01

An antioxidant glycoprotein (Fraction AIV-2) with molecular weight of 27.2 kDa was purified from the ethanol-soluble protein hydrolysate of Mustelus griseus muscle. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of Fraction AIV-2 reached up to 96.73±2.33% and was higher than that of ascorbic acid at the concentration of 5.0mg/mL. Total protein and carbohydrate contents of Fraction AIV-2 were 62.65±0.63% and 33.49±1.60%, respectively. Seventeen amino acids were identified in Fraction AIV-2, most of which are serine. GC-MS analysis showed that Fraction AIV-2 was composed of fucose, arabinose, galactose, glucose and mannose with the ratio of 1.00:1.53:7.27:9.07:2.09. The FT-IR spectrum of Fraction AIV-2 showed typical characteristics of polysaccharide and protein. For Fraction AIV-2, the changes of ultraviolet absorption curve, amino acid composition after the β-elimination reaction and its deglycosylation with the treatment of N-glycosidase F suggested that both O-glycosidic and N-glycosidic bonds were involved in the polysaccharide and protein moieties. Copyright © 2012 Elsevier B.V. All rights reserved.

A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

NASA Astrophysics Data System (ADS)

Santos, Marlus Alves Dos; Teixeira, Francesco Brugnera; Moreira, Heline Hellen Teixeira; Rodrigues, Adele Aud; Machado, Fabrício Castro; Clemente, Tatiana Mordente; Brigido, Paula Cristina; Silva, Rebecca Tavares E.; Purcino, Cecílio; Gomes, Rafael Gonçalves Barbosa; Bahia, Diana; Mortara, Renato Arruda; Munte, Claudia Elisabeth; Horjales, Eduardo; da Silva, Claudio Vieira

2014-03-01

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D 1H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System.

PubMed

Park, Gunhyuk; Oh, Dal-Seok; Kim, Yong-Ung; Park, Moon Ki

2017-01-01

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70. © 2017 S. Karger AG, Basel.

Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.).

PubMed

Park, S H; Strobel, G A

1994-01-05

Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).

Physical-chemical studies of proteins of squid nerve axoplasm, with special reference to the axon fibrous protein.

PubMed

DAVISON, P F; TAYLOR, E W

1960-03-01

The proteins in the axoplasm of the squid, Dosidicus gigas, have been resolved electrophoretically into a major fraction including the fibrous protein, and possibly its structural subunits, and a minor fraction including at least two proteins with low sedimentation coefficients. A partially reversible change in the structure of the fibrous protein occurs under the action of 0.4 M salt or high pH. These experiments have been interpreted to indicate that in the intact fiber one, or a few, protofibrils are arranged helically or longitudinally along the fiber axis, and linked by electrostatic bonds. On the dissociation of these bonds the separated protofibrils assume a less extended form and sediment more rapidly than the intact fibers. Some material with a lower sedimentation rate is also released on the dissociation. This fraction may comprise smaller chain fragments. The volume fraction and the approximate refractive index of the fibers have been calculated.

Physical-Chemical Studies of Proteins of Squid Nerve Axoplasm, with Special Reference to the Axon Fibrous Protein

PubMed Central

Davison, Peter F.; Taylor, Edwin W.

1960-01-01

The proteins in the axoplasm of the squid, Dosidicus gigas, have been resolved electrophoretically into a major fraction including the fibrous protein, and possibly its structural subunits, and a minor fraction including at least two proteins with low sedimentation coefficients. A partially reversible change in the structure of the fibrous protein occurs under the action of 0.4 M salt or high pH. These experiments have been interpreted to indicate that in the intact fiber one, or a few, protofibrils are arranged helically or longitudinally along the fiber axis, and linked by electrostatic bonds. On the dissociation of these bonds the separated protofibrils assume a less extended form and sediment more rapidly than the intact fibers. Some material with a lower sedimentation rate is also released on the dissociation. This fraction may comprise smaller chain fragments. The volume fraction and the approximate refractive index of the fibers have been calculated. PMID:13814536

Anti-angiotensin converting enzyme (ACE) proteins from mycelia of Ganoderma lucidum (Curtis) P. Karst

PubMed Central

2013-01-01

Background Ganoderma lucidum has been purported as a potent remedy in the treatment and prevention of several ailments, including hypertension. This study aimed to explore the anti-ACE potential of protein fractions from the mycelia of G. lucidum. Methods Ganoderma lucidum mycelia were cultivated by submerged fermentation in a liquid medium containing brown sugar and spent brewer’s yeast. Intracellular proteins were fractionated from mycelia crude water extract by ammonium sulphate precipitation, and their angiotensin converting enzyme inhibitory activity was evaluated. The potential anti-ACE protein fractions were further separated by RP-HPLC and characterised using proteomics platforms. Results Preliminary result demonstrated that the mycelia crude water extract inhibited ACE at IC50 value of 1.134 ± 0.036 mg/mL. Following protein fractionation and HPLC purification, the presence of highly potential anti-ACE proteins with the IC50 values less than 200 μg/mL was detected. Characterisation of these proteins demonstrated the presence of four different antihypertensive-related proteins involved in the regulation of blood pressure through different mechanisms. Conclusions This study suggests that the mycelia of G. lucidum has high potential in lowering blood pressure level due to the presence of several antihypertensive-related proteins such as cystathionine beta synthase-like protein, DEAD/DEAH box helicase-like protein, paxillin-like protein, and alpha/beta hydrolase-like protein. PMID:24093919

Quantification of chitinase and thaumatin-like proteins in grape juices and wines.

PubMed

Le Bourse, D; Conreux, A; Villaume, S; Lameiras, P; Nuzillard, J-M; Jeandet, P

2011-09-01

Chitinases and thaumatin-like proteins are important grape proteins as they have a great influence on wine quality. The quantification of these proteins in grape juices and wines, along with their purification, is therefore crucial to study their intrinsic characteristics and the exact role they play in wines. The main isoforms of these two proteins from Chardonnay grape juice were thus purified by liquid chromatography. Two fast protein liquid chromatography (FLPC) steps allowed the fractionation and purification of the juice proteins, using cation exchange and hydrophobic interaction media. A further high-performance liquid chromatography (HPLC) step was used to achieve higher purity levels. Fraction assessment was achieved by mass spectrometry. Fraction purity was determined by HPLC to detect the presence of protein contaminants, and by nuclear magnetic resonance (NMR) spectroscopy to detect the presence of organic contaminants. Once pure fractions of lyophilized chitinase and thaumatin-like protein were obtained, ultra-HPLC (UHPLC) and enzyme-linked immunosorbent assay (ELISA) calibration curves were constructed. The quantification of these proteins in different grape juice and wine samples was thus achieved for the first time with both techniques through comparison with the purified protein calibration curve. UHPLC and ELISA showed very consistent results (less than 16% deviation for both proteins) and either could be considered to provide an accurate and reliable quantification of proteins in the oenology field.

Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen.

PubMed

Plante, Geneviève; Lusignan, Marie-France; Lafleur, Michel; Manjunath, Puttaswamy

2015-08-15

Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general. Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting. Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1). These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

Antiedematogenic and antioxidant properties of high molecular weight protein sub-fraction of Calotropis procera latex in rat.

PubMed

Chaudhary, Priyanka; de Araújo Viana, Carolina; Ramos, Marcio V; Kumar, Vijay L

2015-03-01

The aim was to evaluate the effect of high molecular weight protein fraction of Calotropis procera latex on edema formation and oxidative stress in carrageenan-induced paw inflammation. A sub-plantar injection of carrageenan was given to induce edema in the hind paw of the rat. The inhibitory effect of high molecular weight protein fraction of C. procera latex was evaluated following intravenous administration (5 and 25 mg/kg body weight) and was compared with that of diclofenac given orally (5 mg/kg). The levels of reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and myeloperoxidase (MPO) were measured in the inflamed paw tissue at the end of the study. The high molecular weight protein fraction obtained from the latex of C. procera produced a dose-dependent inhibition of edema formation that was accompanied by normalization of levels of oxidative stress markers (GSH and TBARS) and MPO, a marker for neutrophils in the paw tissue. The high molecular weight protein fraction of C. procera latex ameliorates acute inflammation in the paw through its antioxidant effect.

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism.

PubMed

Stekhoven, Daniel J; Omasits, Ulrich; Quebatte, Maxime; Dehio, Christoph; Ahrens, Christian H

2014-03-17

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled us to distinguish cytoplasmic, peripheral inner membrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion. The work presented here describes the first prokaryotic proteome-wide subcellular localization (SCL) dataset for the emerging pathogen B. henselae (Bhen). The study indicates that suitable subcellular fractionation experiments combined with straight-forward computational analysis approaches assessing the proportion of spectral counts observed in different subcellular fractions are powerful for determining the predominant SCL of a large percentage of the experimentally observed proteins. This includes numerous cases where in silico prediction methods do not provide any prediction. Avoiding a treatment with harsh conditions, cytoplasmic proteins tend to co-fractionate with proteins of the inner membrane fraction, indicative of close functional interactions. The spectral count proportion (SCP) of total membrane versus cytoplasmic fractions allowed us to obtain a good indication about the relative proximity of individual protein complex members to the inner membrane. Using principal component analysis and k-nearest neighbor approaches, we were able to extend the percentage of proteins with a predominant experimental localization to over 90% of all expressed proteins and identified a set of at least 74 outer membrane (OM) proteins. In general, OM proteins represent a rich source of candidates for the development of urgently needed new therapeutics in combat of resurgence of infectious disease and multi-drug resistant bacteria. Finally, by comparing the data from two infection biology relevant conditions, we conceptually explore methods to identify and visualize potential candidates that may partially change their SCL in these different conditions. The data are made available to researchers as a SCL compendium for Bhen and as an assistance in further improving in silico SCL prediction algorithms. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomic characterization of macro-, micro- and nano-extracellular vesicles derived from the same first trimester placenta: relevance for feto-maternal communication.

PubMed

Tong, Mancy; Kleffmann, Torsten; Pradhan, Shantanu; Johansson, Caroline L; DeSousa, Joana; Stone, Peter R; James, Joanna L; Chen, Qi; Chamley, Larry W

2016-04-01

What proteins are carried by extracellular vesicles (EVs) released from normal first trimester placentae? One thousand five hundred and eighty-five, 1656 and 1476 proteins were characterized in macro-, micro- and nano-vesicles, respectively, from first trimester placentae, with all EV fractions being enriched for proteins involved in vesicle transport and inflammation. Placental EVs are being increasingly recognized as important mediators of both healthy and pathological pregnancies. However, current research has focused on detecting changes in specific proteins in particular fractions of vesicles during disease. This is the first study to investigate the full proteome of different-sized fractions of EVs from the same first trimester placenta and highlights the differences/similarities between the vesicle fractions. A well-established ex vivo placental explant culture model was used to generate macro-, micro- and nano-vesicles from 56 first trimester placentae. Vesicle fractions were collected by differential ultracentrifugation, quantified and characterized. Placental macro-, micro- and nano-vesicles were characterized by microscopy, dynamic light scattering and nanoparticle tracking analysis. The proteome of each EV fraction was interrogated using liquid chromatography-coupled tandem mass spectrometry. Results were validated by semi-quantitative western blotting. A total of 1585, 1656 and 1476 proteins were identified in macro-, micro- and nano-vesicles, respectively. One thousand one hundred and twenty-five proteins were shared between all three fractions while up to 223 proteins were unique to each fraction. Gene Ontology pathway analysis showed an enrichment of proteins involved in vesicle transport and inflammation in all three fractions of EVs. The expression levels of proteins involved in internalization of vesicles (annexin V, calreticulin, CD31, CD47), the complement pathway [C3, decay-accelerating factor (DAF), membrane cofactor protein (MCP), protectin] and minor histocompatibility antigens [ATP-dependent RNA helicase (DDX3), ribosomal protein S4 (RPS4)] were different between different-sized EVs. This study is largely hypothesis-generating in nature. It is important to validate these findings using EVs isolated from maternal plasma and the function of the different EV fractions would need further investigation. Our results support the concept that various EV factions can interact with different maternal cells and have unique effects to mediate feto-maternal communication during early pregnancy. This study also provides a list of candidate proteins, which may inform the identification of robust markers that can be used to isolate placental vesicles from the maternal blood in the future. M.T. is a recipient of the University of Auckland Health Research Doctoral Scholarship and the Freemasons Postgraduate Scholarship. This project was supported by a School of Medicine Performance-based research fund (PBRF) grant awarded to L.W.C. No authors have any conflicts of interest to disclose. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cestode parasites release extracellular vesicles with microRNAs and immunodiagnostic protein cargo.

PubMed

Ancarola, María Eugenia; Marcilla, Antonio; Herz, Michaela; Macchiaroli, Natalia; Pérez, Matías; Asurmendi, Sebastián; Brehm, Klaus; Poncini, Carolina; Rosenzvit, Mara; Cucher, Marcela

2017-09-01

Intercellular communication is crucial in multiple aspects of cell biology. This interaction can be mediated by several mechanisms including extracellular vesicle (EV) transfer. EV secretion by parasites has been reported in protozoans, trematodes and nematodes. Here we report that this mechanism is present in three different species of cestodes, Taenia crassiceps, Mesocestoides corti and Echinococcus multilocularis. To confirm this we determined, in vitro, the presence of EVs in culture supernatants by transmission electron microscopy. Interestingly, while T. crassiceps and M. corti metacestodes secrete membranous structures into the culture media, similar vesicles were observed in the interface of the germinal and laminated layers of E. multilocularis metacestodes and were hardly detected in culture supernatants. We then determined the protein cargo in the EV-enriched secreted fractions of T. crassiceps and M. corti conditioned media by LC-MS/MS. Among the identified proteins, eukaryotic vesicle-enriched proteins were identified as expected, but also proteins used for cestode disease diagnosis, proteins related to neurotransmission, lipid binding proteins as well as host immunoglobulins and complement factors. Finally, we confirmed by capillary electrophoresis the presence of intravesicular RNA for both parasites and detected microRNAs by reverse transcription-PCR. This is the first report of EV secretion in cestode parasites and of an RNA secretion mechanism. These findings will provide valuable data not only for basic cestode biology but also for the rational search for new diagnostic targets. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Efficient solubilization of inclusion bodies.

PubMed

Freydell, Esteban J; Ottens, Marcel; Eppink, Michel; van Dedem, Gijs; van der Wielen, Luuk

2007-06-01

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.

Identification of proteinaceous material in the bone of the dinosaur Iguanodon.

PubMed

Embery, Graham; Milner, Angela C; Waddington, Rachel J; Hall, Rachel C; Langley, Martin S; Milan, Anna M

2003-01-01

This study has directed attention at the search for bone-related proteins in an extract of demineralized rib bone of the 120 mya Iguanodon. The inner compact bone was demineralized and the GuCl extract resolved into 11 fractions using anion exchange chromatography, which all contained silver-reactive proteins with various amino acid profiles. Two specific fractions, iv and xi, revealed characteristics typical of contemporary phosphoproteins and proteoglycans, respectively. Fraction iv, 43-57 kDa, contained a high ratio of aspartate and serine, although no phosphate was discernable. Fraction xi contained a band of 41-47 kDa and was rich in chondroitin sulphate and hyaluronan. In addition an early eluting fraction was immunoreactive with an antibody against osteocalcin. A cancellous bone fraction from the same bone sample was also analyzed using N-terminal sequencing and revealed potential similarities with cystatin. While we do not claim to have identified the presence of intact proteins, this study has value in demonstrating that extruded extracellular matrix is protected by its capacity to induce mineralization, which subsequently is important in conserving detectable protein products in ancient skeletal tissues.

Mapping Atheroprotective Functions and Related Proteins/Lipoproteins in Size Fractionated Human Plasma.

PubMed

Swertfeger, Debi K; Li, Hailong; Rebholz, Sandra; Zhu, Xiaoting; Shah, Amy S; Davidson, W Sean; Lu, Long J

2017-04-01

HDL has been shown to possess a variety of cardio-protective functions, including removal of excess cholesterol from the periphery, and inhibition of lipoprotein oxidation. It has been proposed that various HDL subparticles exist, each with distinct protein and lipid compositions, which may be responsible for HDL's many functions. We hypothesized that HDL functions will co-migrate with the operational lipoprotein subspecies when separated by gel filtration chromatography. Plasma from 10 healthy male donors was fractionated and the protein composition of the phospholipid containing fractions was analyzed by mass spectrometry (MS). Each fraction was evaluated for its proteomic content as well as its ability to promote cholesterol efflux and protect low density lipoprotein (LDL) from free radical oxidation. For each function, several peaks of activity were identified across the plasma size gradient. Neither cholesterol efflux or LDL antioxidation activity correlated strongly with any single protein across the fractions. However, we identified multiple proteins that had strong correlations (r values >0.7, p < 0.01) with individual peaks of activity. These proteins fell into diverse functional categories, including those traditionally associated with lipid metabolism, as well as alternative complement cascade, innate immunity and clotting cascades and immunoglobulins. Additionally, the phospholipid and cholesterol concentration of the fractions correlated strongly with cholesterol efflux ( r = 0.95 and 0.82 respectively), whereas the total protein content of the fractions correlated best with antioxidant activity across all fractions ( r = 0.746). Furthermore, two previously postulated subspecies (apoA-I, apoA-II and apoC-1; as well as apoA-I, apoC-I and apoJ) were found to have strong correlations with both cholesterol efflux and antioxidation activity. Up till now, very little has been known about how lipoprotein composition mediates functions like cholesterol efflux and antioxidation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Microbial production of biopolymers from the renewable resource wheat straw.

PubMed

Gasser, E; Ballmann, P; Dröge, S; Bohn, J; König, H

2014-10-01

Production of poly-ß-hydroxybutyrate (PHB) and the chemical basic compound lactate from the agricultural crop 'wheat straw' as a renewable carbon resource. A thermal pressure hydrolysis procedure for the breakdown of wheat straw was applied. By this means, the wheat straw was converted into a partially solubilized hemicellulosic fraction, consisting of sugar monomers, and an insoluble cellulosic fraction, containing cellulose, lignin and a small portion of hemicellulose. The insoluble cellulosic fraction was further hydrolysed by commercial enzymes in monomers. The production of PHB from the sugar monomers originating from hemicellulose or cellulose was achieved by the isolates Bacillus licheniformis IMW KHC 3 and Bacillus megaterium IMW KNaC 2. The basic chemical compound, lactate, a starting compound for the production of polylactide (PLA), was formed by some heterofermentative lactic acid bacteria (LAB) able to grow with xylose from the hemicellulosic wheat straw hydrolysate. Two strains were selected which were able to produce PHB from the sugars both from the hemicellulosic and the cellulosic fraction of the wheat straw. In addition, some of the LAB tested were capable of producing lactate from the hemicellulosic hydrolysate. The renewable resource wheat straw could serve as a substrate for microbiologically produced basic chemicals and biodegradable plastics. © 2014 The Society for Applied Microbiology.

Characterization of soluble microbial products in a drinking water biological aerated filter.

PubMed

Kang, Jia; Ma, Teng-Fei; Zhang, Peng; Gao, Xu; Chen, You-Peng

2016-05-01

Utilization-associated products (UAPs) and biomass-associated products (BAPs) were quantified separately in this study to characterize soluble microbial products (SMPs) in a drinking water lab-scale biological aerated filter (BAF), and their basic characteristics were explored using gel filtration chromatography and three-dimensional excitation-emission matrix (3D-EEM) spectrophotometry with fluorescence regional integration analysis and parallel factor model. UAPs were observed increased with the increase of filter media depth and accumulated after BAF treatment, whereas BAPs were basically constant. 3D-EEM spectroscopy analysis result showed that tryptophan and protein-like compounds were the main components of UAPs and BAPs, and fulvic-acid-like substance was a major component of BAPs, rather than UAPs. In terms of molecular weight (MW) distribution, UAP MW presented a bimodal distribution in the range of 1-5 and >10 kDa, while BAP MW exhibited unimodal distribution with MW >20 kDa fraction accounting for more than 90 %. The macromolecules of UAPs accumulated after BAF treatment. This study provides theoretical support for in-depth study of SMP characteristics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Seema; Simpson, David C.; Tolic, Nikola

We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction.more » Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.« less

Protein Solubility, Digestibility and Fractionation after Germination of Sorghum Varieties

PubMed Central

Afify, Abd El-Moneim M. R.; El-Beltagi, Hossam S.; Abd El-Salam, Samiha M.; Omran, Azza A.

2012-01-01

The changes in crude protein, free amino acids, amino acid composition, protein solubility, protein fractionation and protein digestibility after germination of sorghum were investigated. Sorghum varieties (Dorado, Shandaweel-6, Giza-15) were soaked for 20 h followed by germination for 72 h; the results revealed that crude protein and free amino acids in raw sorghum varieties ranged from 10.62 to 12.46% and 0.66 to 1.03 mg/g, respectively. Shandaweel-6 was the highest variety in crude protein and free amino acids content. After germination, crude protein was decreased and free amino acids were increased. There was an increase in content of valine and phenylalanine amino acids after germination. On the other hand, there was a decrease in most of amino acids after germination. After germination protein solubility was significantly increased. Regarding protein fractions, there was an increase in albumin, globulin and kafirin proteins and a decrease in cross linked kafirin and cross linked glutelin after germination. PMID:22319611

Further studies on the preparation of the immunosuppressive alpha2 protein fraction from serum and its assay in mice

PubMed Central

Mowbray, J. F.; Hargrave, D. C.

1966-01-01

The method for the assay in mice of the α2 glycoprotein fraction of serum with immunosuppressive activity is described. The crude fraction has been shown to be effective in mice only with intravenous injection. When an antigen which is rapidly cleared from the blood is given, a single injection of the protein fraction given 10–20 hours before the exposure to antigen will inhibit the immune response in most animals. Time intervals outside this range are associated with a diminished or absent immunosuppressive effect. The protein fraction does not show activity immediately after preparation, or after reconstitution of freeze-dried material. Within 40 days, however, the activity is restored by storage alone, either frozen or at 4°. The presence of ribonuclease in the fraction is demonstrated, and its possible significance in relation to the immunosuppressive effect is discussed. ImagesFIG. 1 PMID:5926064

Vanishing amplitude of backbone dynamics causes a true protein dynamical transition: H2 NMR studies on perdeuterated C-phycocyanin

NASA Astrophysics Data System (ADS)

Kämpf, Kerstin; Kremmling, Beke; Vogel, Michael

2014-03-01

Using a combination of H2 nuclear magnetic resonance (NMR) methods, we study internal rotational dynamics of the perdeuterated protein C-phycocyanin (CPC) in dry and hydrated states over broad temperature and dynamic ranges with high angular resolution. Separating H2 NMR signals from methyl deuterons, we show that basically all backbone deuterons exhibit highly restricted motion occurring on time scales faster than microseconds. The amplitude of this motion increases when a hydration shell exists, while it decreases upon cooling and vanishes near 175 K. We conclude that the vanishing of the highly restricted motion marks a dynamical transition, which is independent of the time window and of a fundamental importance. This conclusion is supported by results from experimental and computational studies of the proteins myoglobin and elastin. In particular, we argue based on findings in molecular dynamics simulations that the behavior of the highly restricted motion of proteins at the dynamical transition resembles that of a characteristic secondary relaxation of liquids at the glass transition, namely the nearly constant loss. Furthermore, H2 NMR studies on perdeuterated CPC reveal that, in addition to highly restricted motion, small fractions of backbone segments exhibit weakly restricted dynamics when temperature and hydration are sufficiently high.

Proteomic analysis of processing by-products from canned and fresh tuna: identification of potentially functional food proteins.

PubMed

Sanmartín, Esther; Arboleya, Juan Carlos; Iloro, Ibon; Escuredo, Kepa; Elortza, Felix; Moreno, F Javier

2012-09-15

Proteomic approaches have been used to identify the main proteins present in processing by-products generated by the canning tuna-industry, as well as in by-products derived from filleting of skeletal red muscle of fresh tuna. Following fractionation by using an ammonium sulphate precipitation method, three proteins (tropomyosin, haemoglobin and the stress-shock protein ubiquitin) were identified in the highly heterogeneous and heat-treated material discarded by the canning-industry. Additionally, this fractionation method was successful to obtain tropomyosin of high purity from the heterogeneous starting material. By-products from skeletal red muscle of fresh tuna were efficiently fractionated to sarcoplasmic and myofibrillar fractions, prior to the identification based mainly on the combined searching of the peptide mass fingerprint (MALDI-TOF) and peptide fragment fingerprinting (MALDI LIFT-TOF/TOF) spectra of fifteen bands separated by 1D SDS-PAGE. Thus, the sarcoplasmic fraction contained myoglobin and several enzymes that are essential for efficient energy production, whereas the myofibrillar fraction had important contractile proteins, such as actin, tropomyosin, myosin or an isoform of the enzyme creatine kinase. Application of proteomic technologies has revealed new knowledge on the composition of important by-products from tuna species, enabling a better evaluation of their potential applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

High-shear, jet-cooking, and alkali treatment of corn distillers' dried grains to obtain products with enhanced protein, oil and phenolic antioxidants.

PubMed

Inglett, G E; Chen, D; Rose, D J; Berhow, M

2010-08-01

Distillers dried grains (DDG) have potential to be a nutritionally important source of protein, oil and phenolic antioxidants. DDG was subjected to high-shear and jet-cooking, with or without alkaline pH adjustment and autoclaving. Soluble and insoluble fractions were analyzed for protein, oil and ash. Extracts were analyzed for phenolic acids and antioxidant activity. Protein contents were significantly elevated in the insoluble fractions after treatment and the oil content was drastically increased in the insoluble fraction after high-shear and jet-cooking without pH adjustment. Alkaline pH adjustment resulted in a soluble fraction that was highest in phenolic acids, but not antioxidant activity. The highest antioxidant activity was found in the 50% ethanol extract from DDG that had been subjected to high-shear and jet-cooking. These results suggest that high-shear and jet-cooking may be useful processing treatments to increase the value of DDG by producing fractions high in protein, oil and extractable phenolic acids with high antioxidant activity. The DDG fractions and extracts described herein may be useful as food and nutraceutical ingredients, and, if used for these applications, will increase the value of DDG and ease economic burdens on ethanol producers, allowing them to compete in the bio-fuel marketplace.

Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

PubMed

Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

2015-11-06

Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

Navy Bean Flour Particle Size and Protein Content Affect Cake Baking and Batter Quality(1).

PubMed

Singh, Mukti; Byars, Jeffrey A; Liu, Sean X

2015-06-01

Whole navy bean flour and its fine and coarse particle size fractions were used to completely replace wheat flour in cakes. Replacement of wheat flour with whole bean flour significantly increased the protein content. The protein content was adjusted to 3 levels with navy bean starch. The effect of navy bean flour and its fractions at 3 levels of protein on cake batter rheology and cake quality was studied and compared with wheat flour samples. Batters prepared from navy bean flour and its fractions had higher viscosity than the cake flour. Reducing the protein content by addition of starch significantly lowered the viscosity of cake batters. The whole navy bean flour and coarse bean fraction cakes were softer than cakes made with wheat flour but had reduced springiness. Principal component analysis showed a clear discrimination of cakes according to protein. It also showed that low protein navy bean flour cakes were similar to wheat flour cakes. Navy bean flour with protein content adjusted to the level of cake (wheat) flour has potential as a healthy alternative in gluten-free cakes. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Gluten protein composition in several fractions obtained by shear induced separation of wheat flour.

PubMed

van der Zalm, Elizabeth E J; Grabowska, Katarzyna J; Strubel, Maurice; van der Goot, Atze J; Hamer, Rob J; Boom, Remko M

2010-10-13

Recently, it was found that applying curvilinear shear flow in a cone-cone shearing device to wheat flour dough induces separation, resulting in a gluten-enriched fraction in the apex of the cone and gluten-depleted fraction at the outer part. This article describes whether fractionation of the various proteineous components occurs during and after separation of Soissons wheat flour. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high performance liquid chromatography (SE-HPLC) were found to be suitable techniques for this. It is concluded that all protein fractions migrate to the center of the cone as a result of which the composition of the gluten-enriched fraction remains rather similar to that in the original flour. However, the larger glutenin polymer fraction migrated faster, as a result of which the concentration of large polymers was increased with a factor 2.4 compared to that of Soissons flour. The concentration of monomers in the gluten-enriched fraction was decreased to 70% of the original concentration in the original wheat flour.

Carbon turnover in the water-soluble protein of the adult human lens

PubMed Central

Stewart, Daniel N.; Lango, Jozsef; Nambiar, Krishnan P.; Falso, Miranda J. S.; FitzGerald, Paul G.; Rocke, David M.; Hammock, Bruce D.

2013-01-01

Purpose Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the 14C bomb pulse. Methods Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The 14C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the 14C bomb pulse. Results The water-insoluble fractions possessed 14C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction. Conclusions As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract. PMID:23441119

Relationship of cooked rice nutritionally-important starch fractions with other physicochemical properties.

USDA-ARS?s Scientific Manuscript database

Sixteen rice cultivars representing 5 cytosine-thymine repeat (CTn) microsatellite genetic marker groups were analyzed for their cooked rice nutritionally-important starch fractions (rapidly digestible, slowly digestible, and resistant starch), basic grain quality indices (apparent amylose, crude pr...

Effect of flour particle size on microstructural, rheological and physico-sensory characteristics of bread and south Indian parotta.

PubMed

Sakhare, Suresh D; Inamdar, Aashitosh A; Soumya, C; Indrani, D; Rao, G Venkateswara

2014-12-01

Wheat flour fractioned by sieving into four different particle size fractions namely finer fractions (<75 and 75-118 μm), coarser fractions (118-150 and >150 μm) were analyzed for their chemical, rheological, bread & parotta making characteristics. The finer fractions had lower ash, higher dry gluten, damaged starch and sodium dodecysulphate (SDS)-sedimentation value than the coarser fractions. The flour from finer fractions gave bread with best sensory and textural attributes. The parottas from finer fractions showed significantly higher sensory scores for colour, texture, layers, mouthfeel and overall quality score than the coarser fractions. In the micrograph of finer flour fractions, higher number of loosened single starch granules than the aggregates of starch and protein matrix were seen as compared to coarser fractions. These studies indicate that the flour from the finer fractions produce higher quality bread, parotta owing to the presence of higher damaged starch content, quantity and quality of protein in these fractions than coarser fractions.

Secondary Structure and Subunit Composition of Soy Protein In Vitro Digested by Pepsin and Its Relation with Digestibility

PubMed Central

Yang, Yong; Wang, Zhongjiang; Wang, Rui; Sui, Xiaonan; Qi, Baokun; Han, Feifei; Li, Yang; Jiang, Lianzhou

2016-01-01

In the present study, in vitro digestibility and structure of soybean protein isolates (SPIs) prepared from five soybean varieties were investigated in simulated gastric fluid (SGF), using FT-IR microspectroscopy and SDS-PAGE. The result indicated that β-conformations were prone to be hydrolyzed by pepsin preferentially and transformed to unordered structure during in vitro digestion, followed by the digestion of α-helix and unordered structure. A negative linear correlation coefficient was found between the β-conformation contents of five SPIs and their in vitro digestibility values. The intensities of the protein bands corresponding to 7S and 11S fractions were decreased and many peptide bands appeared at 11~15 kDa during enzymatic hydrolysis. β-conglycinin was poorly hydrolyzed with pepsin, especially the β-7S subunit. On the other hand, basic polypeptides of glycinin degraded slower than acidic polypeptides and represented a large proportion of the residual protein after digestion. 11S-A3 of all SPIs disappeared after 1 h digestion. Moreover, a significant negative linear correlation coefficient (r = −0.89) was found between the β-7S contents of five SPIs and their in vitro digestibility values. These results are useful for further studies of the functional properties and bioactive properties of these varieties and laid theoretical foundations for the development of the specific functional soy protein isolate. PMID:27298825

Solid-phase extraction method for the isolation of plant thionins from European mistletoe, wheat and barley using zirconium silicate embedded in poly(styrene-co-divinylbenzene) hollow-monoliths.

PubMed

Hussain, Shah; Güzel, Yüksel; Schönbichler, Stefan A; Rainer, Matthias; Huck, Christian W; Bonn, Günther K

2013-09-01

Thionins are cysteine-rich, biologically active small (∼5 kDa) and basic proteins occurring ubiquitously in the plant kingdom. This study describes an efficient solid-phase extraction (SPE) method for the selective isolation of these pharmacologically active proteins. Hollow-monolithic extraction tips based on poly(styrene-co-divinylbenzene) with embedded zirconium silicate nano-powder were designed, which showed an excellent selectivity for sulphur-rich proteins owing to strong co-ordination between zirconium and the sulphur atoms from the thiol-group of cysteine. The sorbent provides a combination of strong hydrophobic and electrostatic interactions which may help in targeted separation of certain classes of proteins in a complex mixture based upon the binding strength of different proteins. European mistletoe, wheat and barley samples were used for selective isolation of viscotoxins, purothionins and hordothionins, respectively. The enriched fractions were subjected to analysis by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometer to prove the selectivity of the SPE method towards thionins. For peptide mass-fingerprint analysis, tryptic digests of SPE eluates were examined. Reversed-phase high-performance liquid chromatography hyphenated to diode-array detection was employed for the purification of individual isoforms. The developed method was found to be highly specific for the isolation and purification of thionins.

The relationships between the isoelectric point and: length of proteins, taxonomy and ecology of organisms

PubMed Central

Kiraga, Joanna; Mackiewicz, Pawel; Mackiewicz, Dorota; Kowalczuk, Maria; Biecek, Przemysław; Polak, Natalia; Smolarczyk, Kamila; Dudek, Miroslaw R; Cebrat, Stanislaw

2007-01-01

Background The distribution of isoelectric point (pI) of proteins in a proteome is universal for all organisms. It is bimodal dividing the proteome into two sets of acidic and basic proteins. Different species however have different abundance of acidic and basic proteins that may be correlated with taxonomy, subcellular localization, ecological niche of organisms and proteome size. Results We have analysed 1784 proteomes encoded by chromosomes of Archaea, Bacteria, Eukaryota, and also mitochondria, plastids, prokaryotic plasmids, phages and viruses. We have found significant correlation in more than 95% of proteomes between the protein length and pI in proteomes – positive for acidic proteins and negative for the basic ones. Plastids, viruses and plasmids encode more basic proteomes while chromosomes of Archaea, Bacteria, Eukaryota, mitochondria and phages more acidic ones. Mitochondrial proteomes of Viridiplantae, Protista and Fungi are more basic than Metazoa. It results from the presence of basic proteins in the former proteomes and their absence from the latter ones and is related with reduction of metazoan genomes. Significant correlation was found between the pI bias of proteomes encoded by prokaryotic chromosomes and proteomes encoded by plasmids but there is no correlation between eukaryotic nuclear-coded proteomes and proteomes encoded by organelles. Detailed analyses of prokaryotic proteomes showed significant relationships between pI distribution and habitat, relation to the host cell and salinity of the environment, but no significant correlation with oxygen and temperature requirements. The salinity is positively correlated with acidicity of proteomes. Host-associated organisms and especially intracellular species have more basic proteomes than free-living ones. The higher rate of mutations accumulation in the intracellular parasites and endosymbionts is responsible for the basicity of their tiny proteomes that explains the observed positive correlation between the decrease of genome size and the increase of basicity of proteomes. The results indicate that even conserved proteins subjected to strong selectional constraints follow the global trend in the pI distribution. Conclusion The distribution of pI of proteins in proteomes shows clear relationships with length of proteins, subcellular localization, taxonomy and ecology of organisms. The distribution is also strongly affected by mutational pressure especially in intracellular organisms. PMID:17565672

Fractionation of the Gulf toadfish intestinal precipitate organic matrix reveals potential functions of individual proteins.

PubMed

Schauer, Kevin L; Grosell, Martin

2017-06-01

The regulatory mechanisms behind the production of CaCO 3 in the marine teleost intestine are poorly studied despite being essential for osmoregulation and responsible for a conservatively estimated 3-15% of annual oceanic CaCO 3 production. It has recently been reported that the intestinally derived precipitates produced by fish as a byproduct of their osmoregulatory strategy form in conjunction with a proteinaceous matrix containing nearly 150 unique proteins. The individual functions of these proteins have not been the subject of investigation until now. Here, organic matrix was extracted from precipitates produced by Gulf toadfish (Opsanus beta) and the matrix proteins were fractionated by their charge using strong anion exchange chromatography. The precipitation regulatory abilities of the individual fractions were then analyzed using a recently developed in vitro calcification assay, and the protein constituents of each fraction were determined by mass spectrometry. The different fractions were found to have differing effects on both the rate of carbonate mineral production, as well as the morphology of the crystals that form. Using data collected from the calcification assay as well as the mass spectrometry experiments, individual calcification promotional indices were calculated for each protein, giving the first insight into the functions each of these matrix proteins may play in regulating precipitation. Copyright © 2017 Elsevier Inc. All rights reserved.

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve an 89K protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, S.K.; Woda, B.

1986-03-01

Membrane immunoglobulin of B-lymphocytes is thought to play an important role in antigen recognition and cellular activation. Binding of cross-linking ligands to surface immunoglobulin (SIg) on intact cells converts it to a detergent insoluble state, and this conversion is associated with the transmission of a mitogenic signal. Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons in buffers which convert F-actin to G-actin ((Buffer 1), 0.34M sucrose, 0.5mM ATP, 0.5mM Dithiothrietol and lmM EDTA). Immunoprecipitation of SIg from the detergent soluble fraction of /sup 35/S-methionine labeled non ligand treated rat B-cells results in the co-isolation of anmore » 89K protein and a 44K protein, presumably actin. The 89K protein is not associated with the fraction of endogenous detergent insoluble SIg. On treatment of rat B cells with cross-linking ligand (anti-Ig) the 89K protein becomes detergent insoluble along with most of the SIg and co-isolates with SIg on immunoprecipitation of the detergent insoluble, buffer l solubilized fraction. The migration of the SIg-associated 89K protein from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that this protein might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of a mitogenic signal.« less

[Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin].

PubMed

Durliat, M

1983-11-01

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important rôle in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Workplace Math I: Easing into Math.

ERIC Educational Resources Information Center

Wilson, Nancy; Goschen, Claire

This basic skills learning module includes instruction in performing basic computations, using general numerical concepts such as whole numbers, fractions, decimals, averages, ratios, proportions, percentages, and equivalents in practical situations. The problems are relevant to all aspects of the printing and manufacturing industry, with emphasis…

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

PubMed

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.

Biochemical Changes in Terminal Root Galls Caused by an Ectoparasitic Nematode, Longidorus africanus: Amino Acids.

PubMed

Epstein, E; Cohn, E

1971-10-01

The amino acids of terminal root galls caused by Longidorus africanus on bur marigold (Bidens tripartita L.) and grapevine (Vitis vinifera L.) were studied. The galled roots of bur marigold contained 73% more cell-wall protein and 184% more free amino acids. The main changes among the free amino acids of the galled tissue were a large increase (1900%) in proline and a decrease in aspartic acid (56%) compared with the respective check tissue. Hydroxyproline decreased in the wall protein fraction from 5.6% in the healthy tissue to 3.6% in the infected tissue.Percent of hydroxyproline in total amino acids of the wall protein fraction of grapevine roots decreased from 0.7% in the healthy tissue to 0.3% in the galled tissue, and total proteins of this fraction decreased from 9.5 mg to 4.5 rag, respectively. Total protein in the protoplasmic fraction also decreased from 3.0 mg in healthy to 1.0 mg in infected roots. No change was noticed in total proteins in the free amino acids fraction but free proline decreased 40% in the infected roots.The relationship of these differences to the specific reactions of the hosts to nematode feeding is discussed.

Biochemical and functional characterization of Parawixia bistriata spider venom with potential proteolytic and larvicidal activities.

PubMed

Gimenez, Gizeli S; Coutinho-Neto, Antonio; Kayano, Anderson M; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Stábeli, Rodrigo G

2014-01-01

Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications.

Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

PubMed Central

Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

2014-01-01

Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

21 CFR 640.92 - Tests on final product.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.92 Tests on...) Heat stability. A final container sample of Plasma Protein Fraction (Human) shall remain unchanged, as...

21 CFR 640.92 - Tests on final product.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.92 Tests on...) Heat stability. A final container sample of Plasma Protein Fraction (Human) shall remain unchanged, as...

21 CFR 640.92 - Tests on final product.

Code of Federal Regulations, 2011 CFR

2011-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.92 Tests on...) Heat stability. A final container sample of Plasma Protein Fraction (Human) shall remain unchanged, as...

21 CFR 640.92 - Tests on final product.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.92 Tests on...) Heat stability. A final container sample of Plasma Protein Fraction (Human) shall remain unchanged, as...

21 CFR 640.92 - Tests on final product.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.92 Tests on...) Heat stability. A final container sample of Plasma Protein Fraction (Human) shall remain unchanged, as...

Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

PubMed

Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

2016-10-10

The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry.

PubMed

Griaud, François; Denefeld, Blandine; Lang, Manuel; Hensinger, Héloïse; Haberl, Peter; Berg, Matthias

2017-07-01

Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.

Methylation of yeast ribosomal protein Rpl3 promotes translational elongation fidelity.

PubMed

Al-Hadid, Qais; Roy, Kevin; Chanfreau, Guillaume; Clarke, Steven G

2016-04-01

Rpl3, a highly conserved ribosomal protein, is methylated at histidine 243 by the Hpm1 methyltransferase in Saccharomyces cerevisiae. Histidine 243 lies close to the peptidyl transferase center in a functionally important region of Rpl3 designated as the basic thumb that coordinates the decoding, peptidyl transfer, and translocation steps of translation elongation. Hpm1 was recently implicated in ribosome biogenesis and translation. However, the biological role of methylation of its Rpl3 substrate has not been identified. Here we interrogate the role of Rpl3 methylation at H243 by investigating the functional impact of mutating this histidine residue to alanine (rpl3-H243A). Akin to Hpm1-deficient cells, rpl3-H243A cells accumulate 35S and 23S pre-rRNA precursors to a similar extent, confirming an important role for histidine methylation in pre-rRNA processing. In contrast, Hpm1-deficient cells but not rpl3-H243A mutants show perturbed levels of ribosomal subunits. We show that Hpm1 has multiple substrates in different subcellular fractions, suggesting that methylation of proteins other than Rpl3 may be important for controlling ribosomal subunit levels. Finally, translational fidelity assays demonstrate that like Hpm1-deficient cells, rpl3-H243A mutants have defects in translation elongation resulting in decreased translational accuracy. These data suggest that Rpl3 methylation at H243 is playing a significant role in translation elongation, likely via the basic thumb, but has little impact on ribosomal subunit levels. Hpm1 is therefore a multifunctional methyltransferase with independent roles in ribosome biogenesis and translation. © 2016 Al-Hadid et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan

2010-11-26

Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In thismore » study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular sensitivity to anticancer treatments. These findings may provide pivotal insights in the context of fractionated radiation-based therapeutic interventions in brain cancer.« less

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirako, Yoshiaki, E-mail: s47526a@cc.nagoya-u.ac.jp; Yonemoto, Yuki; Yamauchi, Tomoe

2014-06-10

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and depositedmore » extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. - Highlights: • A defined condition promoted accumulation of hemidesmosomes in human cultured cells. • A fraction isolated from the cells contained eight major polypeptides. • The polypeptides were the five major hemidesmosome proteins and laminin-332. • The cultured cells deposited laminin-332 in its unprocessed form under the condition. • We report a method to prepare a fraction highly enriched in hemidesmosome proteins.« less

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

PubMed

Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

Single-step isolation of extracellular vesicles by size-exclusion chromatography

PubMed Central

Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

2014-01-01

Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. PMID:25279113

Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria.

PubMed

McLean, Richard; Inglis, G Douglas; Mosimann, Steven C; Uwiera, Richard R E; Abbott, D Wade

2017-01-01

Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

Compositional Changes and Baking Performance of Rye Dough As Affected by Microbial Transglutaminase and Xylanase.

PubMed

Grossmann, Isabel; Döring, Clemens; Jekle, Mario; Becker, Thomas; Koehler, Peter

2016-07-20

Doughs supplemented with endoxylanase (XYL) and varying amounts of microbial transglutaminase (TG) were analyzed by sequential protein extraction, quantitation of protein fractions and protein types, and determination of water-extractable arabinoxylans. With increasing TG activity, the concentration of prolamins and glutelins decreased and increased, respectively, and the prolamin-to-glutelin ratio strongly declined. The overall amount of extractable protein decreased with increasing TG level showing that cross-linking by TG provided high-molecular-weight protein aggregates. The decrease of the high-molecular-weight arabinoxylan fraction and the concurrent increase of the medium-molecular-weight fraction confirmed the degradation of arabinoxylans by XYL. However, XYL addition did not lead to significant improved cross-linking of rye proteins by TG. Volume and crumb hardness measurements of bread showed increased protein connectivity induced by XYL and TG. Significant positive effects on the final bread quality were especially obtained by XYL addition.

Effect of Protein Molecular Weight Distribution on Kernel and Baking Characteristics and Intra-varietal Variation in Hard Spring Wheats

USDA-ARS?s Scientific Manuscript database

Specific wheat protein fractions are known to have distinct associations with wheat quality traits. Research was conducted on 10 hard spring wheat cultivars grown at two North Dakota locations to identify protein fractions that affected wheat kernel characteristics and breadmaking quality. SDS ext...

Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

PubMed

Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

2009-12-01

The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

Lens protein composition, glycation and high molecular weight aggregation in aging rats.

PubMed

Swamy, M S; Abraham, E C

1987-10-01

Because of minimal or no turnover, lens proteins are subjected to substantial post-translational modifications which in turn disrupt lens architecture and change the optical properties leading to senile cataract formation. Progressive glycation is believed to have the potential to initiate the changes that are conducive to lens opacification. Fisher 344 rats were systematically followed from juvenile to older and aged phases of their life to study the relationship between lens glycation and high molecular weight (HMW) aggregate formation as well as quantitative and qualitative changes in lens crystallins. Levels of glycated proteins were quantified by affinity chromatography. Changes in lens crystallin composition and HMW aggregate formation were monitored by molecular sieve HPLC, further confirmed by SDS-PAGE and IEF techniques. As the age advances HMW and insoluble proteins increase with a concomitant disappearance of gamma-crystallins from soluble fraction. This disappearance of gamma-crystallins coincided with increased glycation (approximately 2-fold higher in insoluble fraction) and decreased sulfhydryl groups from soluble fraction. It appears that lens protein glycation, disappearance of gamma-crystallins and sulfhydryls from soluble fraction and increase of insoluble fraction and HMW aggregate are interrelated.

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins.

PubMed

Jacob, Jaison; Louis, John M; Nesheiwat, Issa; Torchia, Dennis A

2002-11-01

Analysis of 2D [(13)C,(1)H]-HSQC spectra of biosynthetic fractionally (13)C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional (13)C labeling yields aromatic rings in which some of the (13)C-(13)C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the delta-, epsilon- and zeta-carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the (13)C constant-time period in 2D [(13)C,(1)H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic (13)C CSA and (13)C-(1)H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution (13)C constant-time spectra with good sensitivity.

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

PubMed Central

Anklesaria, Jenifer H.; Jagtap, Dhanashree D.; Pathak, Bhakti R.; Kadam, Kaushiki M.; Joseph, Shaini; Mahale, Smita D.

2013-01-01

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma. PMID:23469287

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

PubMed

Dash, Priyanka; Ghosh, Goutam

2017-12-01

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

Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

PubMed Central

2006-01-01

Abstract Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis. PMID:16548331

Differential antigenic protein recovery from Taenia solium cyst tissues using several detergents.

PubMed

Navarrete-Perea, José; Orozco-Ramírez, Rodrigo; Moguel, Bárbara; Sciutto, Edda; Bobes, Raúl J; Laclette, Juan P

2015-07-01

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures. Copyright © 2015 Elsevier B.V. All rights reserved.

Unraveling sterol-dependent membrane phenotypes by analysis of protein abundance-ratio distributions in different membrane fractions under biochemical and endogenous sterol depletion.

PubMed

Zauber, Henrik; Szymanski, Witold; Schulze, Waltraud X

2013-12-01

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion*

PubMed Central

Zauber, Henrik; Szymanski, Witold; Schulze, Waltraud X.

2013-01-01

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions. PMID:24030099

Alterations in the lenticular protein profile in experimental selenite-induced cataractogenesis and prevention by ellagic acid.

PubMed

Sakthivel, Muniyan; Geraldine, Pitchairaj; Thomas, Philip A

2011-08-01

Accumulating evidence suggests that oxidative stress underlies age-related formation of cataract, and that antioxidants retard cataractogenesis. This study aimed to evaluate whether ellagic acid, a natural polyphenol with antioxidant properties, prevents alterations in the lenticular protein profile in an experimental model of selenite cataract. Alterations in lenticular protein were determined by two-dimensional electrophoresis (2DE) and image analysis. Eluted αA-crystallin spots were analyzed by mass spectrometry. Western blot analysis was also performed to confirm the differential expression of certain crystallins and cytoskeletal proteins. In cataractous lenses, 2DE and image analysis revealed approximately 45 and 60 prominent spots in soluble and insoluble protein fractions respectively. Analysis of the pI and molecular weight of protein spots revealed differences in the expression of crystallin proteins in soluble and insoluble fractions. Western blot analysis confirmed changes in the expression of αA- and βB1- crystallins in both soluble and insoluble protein fractions, while mass spectrometry confirmed the degradation of αA-crystallin in selenite cataractous lenses. Western blot analysis also confirmed the occurrence of altered expression of certain cytoskeletal proteins in insoluble fractions. However, the lenticular protein profile in lenses from selenite-challenged, ellagic acid-treated rats was essentially similar to that noted in lenses from normal rats. The present study confirms the importance of structural and cytoskeletal proteins in the maintenance of lenticular transparency; the results also suggest that ellagic acid prevents lenticular protein alterations induced by selenite in an experimental setting.

Basic Math I.

ERIC Educational Resources Information Center

Mercer County Community Coll., Trenton, NJ.

This document offers instructional materials for a 60-hour course on basic math operations involving decimals, fractions, and proportions as applied in the workplace. The course, part of a workplace literacy project developed by Mercer County Community College (New Jersey) and its partners, contains the following: course outline; 17 lesson…

Impact of Carrier Fluid Composition on Recovery of Nanoparticles and Proteins in Flow Field Flow Fractionation

PubMed Central

Schachermeyer, Samantha; Ashby, Jonathan; Kwon, MinJung; Zhong, Wenwan

2012-01-01

Flow field flow fractionation (F4) is an invaluable separation tool for large analytes, including nanoparticles and biomolecule complexes. However, sample loss due to analyte-channel membrane interaction limits extensive usage of F4 at present, which could be strongly affected by the carrier fluid composition. This work studied the impacts of carrier fluid (CF) composition on nanoparticle (NP) recovery in F4, with focus on high ionic strength conditions. Successful analysis of NPs in a biomolecules-friendly environment could expand the applicability of F4 to the developing field of nanobiotechnology. Recovery of the unfunctionalized polystyrene NPs of 199-, 102-, and 45-nm in CFs with various pH (6.2, 7.4 and 8.2), increasing ionic strength (0–0.1 M), and different types of co- and counter-ions, were investigated. Additionally, elution of the 85-nm carboxylate NPs and two proteins, human serum albumin (HSA) and immunoglobulin (IgG), at high ionic strengths (0–0.15 M) was investigated. Our results suggested that; 1) Electrostatic repulsion between the negatively charged NPs and the regenerated cellulose membrane was the main force to avoid particle adsorption on the membrane; 2) Larger particles experienced higher attractive force and thus were influenced more by variation in CF composition; and 3) Buffers containing weak anions or NPs with weak anion as the surface functional groups provided higher tolerance to the increase in ionic strength, owing to more anions being trapped inside the NP porous structure. Protein adsorption onto the membrane was also briefly investigated in salted CFs, using human serum albumin and immunoglobulin. We believe our findings could help to identify the basic carrier fluid composition for higher sample recovery in F4 analysis of nanoparticles in a protein-friendly environment, which will be useful for applying F4 in bioassays and in nanotoxicology studies. PMID:23058938

A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate

PubMed Central

Keren, Leeat; Segal, Eran; Milo, Ron

2016-01-01

Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms. PMID:27073913

The Escherichia coli Peripheral Inner Membrane Proteome*

PubMed Central

Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

2013-01-01

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. PMID:23230279

The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

NASA Technical Reports Server (NTRS)

Solomon, V.; Lecker, S. H.; Goldberg, A. L.

1998-01-01

In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

PubMed Central

Mather, I H; Sullivan, C H; Madara, P J

1982-01-01

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk. Images Fig. 1. Fig. 2. Fig. 3. PMID:7046730

Games for Mathematics Skill Practice.

ERIC Educational Resources Information Center

Ludeman, Clinton; Sevier, Bonnie

1982-01-01

Multivision is designed to practice simple multiplication and division with one-digit numbers, and is played similarly to Sorry. Fraction Monopoly was designed to assist in practicing addition and subtraction skills with fractions, along with recognizing basic parts and matching numerals with pictorial representations, and is similar to Monopoly.…

Preparation of the Nuclear Matrix for Parallel Microscopy and Biochemical Analyses.

PubMed

Wilson, Rosemary H C; Hesketh, Emma L; Coverley, Dawn

2016-01-04

Immobilized proteins within the nucleus are usually identified by treating cells with detergent. The detergent-resistant fraction is often assumed to be chromatin and is described as such in many studies. However, this fraction consists of both chromatin-bound and nuclear-matrix-bound proteins. To investigate nuclear-matrix-bound proteins alone, further separation of these fractions is required; the DNA must be removed so that the remaining proteins can be compared with those from untreated cells. This protocol uses a nonionic detergent (Triton X-100) to remove membranes and soluble proteins from cells under physiologically relevant salt concentrations, followed by extraction with 0.5 m NaCl, digestion with DNase I, and removal of fragmented DNA. It uses a specialized buffer (cytoskeletal buffer) to stabilize the cytoskeleton and nuclear matrix in relatively gentle conditions. Nuclear matrix proteins can then be assessed by either immunofluorescence (IF) and immunoblotting (IB). IB has the advantage of resolving different forms of a protein of interest, and the soluble fractions can be analyzed. The major advantage of IF analysis is that individual cells (rather than homogenized populations) can be monitored, and the spatial arrangement of proteins bound to residual nuclear structures can be revealed. © 2016 Cold Spring Harbor Laboratory Press.

Mutagenicity of nitrogen compounds from synthetic crude oils: collection, separation and biological testing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, T K; Epler, J L; Guerin, M R

1980-01-01

In order to determine the long range health effects such as carcinogenicity/mutagenicity/teratogenicity/toxicity, associated with the newly emerging energy technologies, we have utilized the Ames Salmonella assay to evaluate mutagenic properties of synthetic fuels. Coupling with class fractionation was necessary. Organic extraction and liquid/liquid partitioning was used to separate acidic and basic fraction. The neutral material was separated using Sephadex LH-20 gel filtration into saturated and aromatic fractions of various ring sizes. The alkaline fraction was subfractionated eluting with benzene and ethanol on a basic alumina column and then with isopropanol and acetone using a Sephadex LH-20 gel column. The frameshiftmore » strain TA-98 was utilized along with Aroclor-induced rat liver homogenate (S-9 mix) for the mutagenicity assay. The natural crude oils were slightly mutagenic, the polynucleararomatics constituting the activity, while the coal-derived fuels indicated mutagenicity associated with alkaline constituents as well as polyaromatics. Hydrotreated coal (H-coal, HDT) or Shale (Paraho-Shale oil, HDT) derived fuels were not mutagenic. Ninety percent of the mutagenic activity in alkaline fraction was recovered in the acetone subfraction. High resolution spectroscopy of this fraction indicates polycyclic aromatic primary amines along with azaarenes as organic constituents responsible for the mutagenic activity associated with shale- and coal-derived fuels.« less

Isolation and identification of antioxidant peptides from enzymatically hydrolyzed rice bran protein.

PubMed

Wattanasiritham, Ladda; Theerakulkait, Chockchai; Wickramasekara, Samanthi; Maier, Claudia S; Stevens, Jan F

2016-02-01

Khao Dawk Mali 105 rice bran protein (RBP) was fractionated into albumin (12.5%), globulin (13.9%), glutelin (70.8%) and prolamine (2.9%). The native and denatured RBP fractions were hydrolyzed with papain and trypsin for 3h at optimum conditions. The RBP fractions and their hydrolysates were evaluated for their antioxidant activity by the Oxygen Radical Absorbance Capacity (ORAC) assay. The trypsin-hydrolyzed denatured albumin exhibited the highest antioxidant activity with an ORAC value of 4.07 μmol of Trolox equivalent (TE)/mg protein. This hydrolysate was separated by using RP-HPLC and three fractions with high antioxidant activity were examined by LTQ-FTICR ESI mass spectrometry. The MW of the peptides from these fractions were 800-2100 Da. and consisted of 6-21 amino acid residues. Most of the peptides from the fractions demonstrated typical characteristics of well-known antioxidant peptides. The results suggest that trypsin-hydrolyzed denatured rice bran albumin might be useful as a natural food antioxidant. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation and partial characterization of a protein fraction from the opossum (Didelphis marsupialis) serum, with protecting property against the Bothrops jararaca snake venom.

PubMed

Perales, J; Muñoz, R; Moussatché, H

1986-01-01

Two separated methods were used to purify a fraction from the opossum (Didelphis marsupialis) serum able to protect mice against Bothrops jararaca venom. The first of them included an initial batch DEAE-Cellulose ion-exchange of the serum, followed by another ion-exchange chromatography on a Carboxymethyl Sepharose column. The second method was a column ion-exchange chromatography on DEAE-Sephacel. These techniques allowed to obtain a protein fraction which resulted homogeneous in cellulose acetate and conventional polyacrylamide gel electrophoresis. The obtained protein fraction proved to be a glycoprotein according to the positive staining with periodic acid Schiff. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the B-mercaptoethanol-reduced fraction showed heterogeneity and allowed to estimate molecular weights in the range of 42,000 to 58,000 daltons. The obtained serum fraction could effectively block the lethal effect of B. jararaca venom when jointly injected to laboratory mice by peritoneal route.

Altered levels of acid, basic, and neutral peptidase activity and expression in human clear cell renal cell carcinoma.

PubMed

Varona, Adolfo; Blanco, Lorena; López, José I; Gil, Javier; Agirregoitia, Ekaitz; Irazusta, Jon; Larrinaga, Gorka

2007-02-01

Peptides play important roles in cell regulation and signaling in many tissues and are regulated by peptidases, most of which are highly expressed in the kidney. Several peptide convertases have a function in different tumor stages, and some have been clearly characterized as diagnostic and prognostic markers for solid tumors, including renal cancer; however, little is known about their in vivo role in kidney tumors. The present study compares the activity of a range of peptidases in human tumor samples and nontumor tissue obtained from clear cell renal cell carcinoma (CCRCC) patients. To cover the complete spectrum and subcellular distribution of peptide-converting activity, acid, neutral, basic, and omega activities were selected. CCRCC displays a selective and restricted pattern of peptidase activities. Puromycin-sensitive aminopeptidase activity in the tumor increases [tumor (t) = 10,775 vs. nontumor (n) = 7,635 units of peptidase (UP)/mg protein; P < 0.05], whereas aminopeptidase N decreases (t = 6,664 vs. n = 33,381 UP/mg protein; P < 0.001). Aminopeptidase B activity of the particulate fraction in tumors decreases (t = 2,399 vs. n = 13,536 UP/mg protein; P < 0.001) compared with nontumor tissues, and aspartyl-aminopeptidase activity decreases significantly in CCRCC (t = 137 vs. n = 223 UP/mg protein; P < 0.05). Soluble and particulate pyroglutamyl peptidase I activities, aminopeptidase A activity, and soluble aminopeptidase B activity do not vary in renal cancer. The relative expression for the aforementioned peptidases, assayed using quantitative RT-PCR, increases in CCRCC for aminopeptidases B (1.5-fold) and A (19-fold), aspartyl-aminopeptidase (3.9-fold), puromycin-sensitive aminopeptidase (2.5-fold), and pyroglutamyl peptidase I (7.6-fold). Only aminopeptidase N expression decreases in tumors (1.3-fold). This peptidase activity profile in the neoplastic kidney suggests a specific role for the studied convertases and the possible involvement of an intracrine renin-angiotensin system in the pathogenesis of CCRCC.

Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation

NASA Technical Reports Server (NTRS)

Wing, S. S.; Haas, A. L.; Goldberg, A. L.

1995-01-01

The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

PubMed Central

2010-01-01

Background Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition. PMID:20704702

Changes in topography and function of thylakoid membranes following membrane protein phosphorylation.

PubMed

Black, M T; Lee, P; Horton, P

1986-09-01

Changes in topography and function of pea (Pisum sativum L.) thylakoid membrane fractions following membrane protein phosphorylation have been studied. After protein phosphorylation the stromal membrane fraction had a higher chlorophyll a/b ratio, an increased content of light-harvesting chlorophyll protein and a higher ratio of chlorophyll to cytochrome f. This indicates that a pool of light-harvesting chlorophyll protein migrates from the photosystem II-enriched grana regions to the photosystem I-enriched stroma lamellae, in agreement with Kyle et al. (1984, Biochim. Biophys. Acta 765, 89-96) and Larsson et al. (1983, Eur. J. Biochem. 136, 25-29). Phosphorylation caused a stimulation in the rate of light-limited photosystem-I electron transfer in the unappressed membrane fraction, indicating that the translocated LHC-II becomes functionally associated with photosystem I.

Short communication: Potential of Fresco-style cheese whey as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme inhibitory activities.

PubMed

Tarango-Hernández, S; Alarcón-Rojo, A D; Robles-Sánchez, M; Gutiérrez-Méndez, N; Rodríguez-Figueroa, J C

2015-11-01

Recently, traditional Mexican Fresco-style cheese production has been increasing, and the volume of cheese whey generated represents a problem. In this study, we investigated the chemical composition of Fresco-style cheese wheys and their potential as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme (ACE)-inhibitory activities. Three samples from Fresco, Panela, and Ranchero cheeses whey were physicochemically characterized. Water-soluble extracts were fractionated to obtain whey fractions with different molecular weights: 10-5, 5-3, 3-1 and <1 kDa. The results indicated differences in the lactose, protein, ash, and dry matter contents (% wt/wt) in the different Fresco-style cheese wheys. All whey fractions had antioxidant and ACE-inhibitory activities. The 10-5 kDa whey fraction of Ranchero cheese had the highest Trolox equivalent antioxidant capacity (0.62 ± 0.00 mM), and the 3-1 kDa Panela and Fresco cheese whey fractions showed the highest ACE-inhibitory activity (0.57 ± 0.02 and 0.59 ± 0.04 μg/mL 50%-inhibitory concentration values, respectively). These results suggest that Fresco-style cheese wheys may be a source of protein fractions with bioactivity, and thus could be useful ingredients in the manufacture of functional foods with increased nutritional value. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

PubMed

Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

2014-01-01

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents.

PubMed

Rabani, Vahideh; Davani, Siamak; Gambert-Nicot, Ségolène; Meneveau, Nicolas; Montange, Damien

2016-11-01

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Hypoallergenic and Physicochemical Properties of the A2 β-Casein Fractionof Goat Milk.

PubMed

Jung, Tae-Hwan; Hwang, Hyo-Jeong; Yun, Sung-Seob; Lee, Won-Jae; Kim, Jin-Wook; Ahn, Ji-Yun; Jeon, Woo-Min; Han, Kyoung-Sik

2017-01-01

Goat milk has a protein composition similar to that of breast milk and contains abundant nutrients, but its use in functional foods is rather limited in comparison to milk from other sources. The aim of this study was to prepare a goat A2 β-casein fraction with improved digestibility and hypoallergenic properties. We investigated the optimal conditions for the separation of A2 β-casein fraction from goat milk by pH adjustment to pH 4.4 and treating the casein suspension with calcium chloride (0.05 M for 1 h at 25°C). Selective reduction of β-lactoglobulin and α s -casein was confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. The hypoallergenic property of A2 β-casein fraction was examined by measuring the release of histamine and tumor necrosis factor alpha from HMC-1 human mast cells exposed to different proteins, including A2 β-casein fraction. There was no significant difference in levels of both indicators between A2 β-casein treatment and the control (no protein treatment). The A2 β-casein fraction is abundant in essential amino acids, especially, branched-chain amino acids (leucine, valine, and isoleucine). The physicochemical properties of A2 β-casein fraction, including protein solubility and viscosity, are similar to those of bovine whole casein which is widely used as a protein source in various foods. Therefore, the goat A2 β-casein fraction may be useful as a food material with good digestibility and hypoallergenic properties for infants, the elderly, and people with metabolic disorders.

ComplexQuant: high-throughput computational pipeline for the global quantitative analysis of endogenous soluble protein complexes using high resolution protein HPLC and precision label-free LC/MS/MS.

PubMed

Wan, Cuihong; Liu, Jian; Fong, Vincent; Lugowski, Andrew; Stoilova, Snejana; Bethune-Waddell, Dylan; Borgeson, Blake; Havugimana, Pierre C; Marcotte, Edward M; Emili, Andrew

2013-04-09

The experimental isolation and characterization of stable multi-protein complexes are essential to understanding the molecular systems biology of a cell. To this end, we have developed a high-throughput proteomic platform for the systematic identification of native protein complexes based on extensive fractionation of soluble protein extracts by multi-bed ion exchange high performance liquid chromatography (IEX-HPLC) combined with exhaustive label-free LC/MS/MS shotgun profiling. To support these studies, we have built a companion data analysis software pipeline, termed ComplexQuant. Proteins present in the hundreds of fractions typically collected per experiment are first identified by exhaustively interrogating MS/MS spectra using multiple database search engines within an integrative probabilistic framework, while accounting for possible post-translation modifications. Protein abundance is then measured across the fractions based on normalized total spectral counts and precursor ion intensities using a dedicated tool, PepQuant. This analysis allows co-complex membership to be inferred based on the similarity of extracted protein co-elution profiles. Each computational step has been optimized for processing large-scale biochemical fractionation datasets, and the reliability of the integrated pipeline has been benchmarked extensively. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Math 3013--Developmental Mathematics I and II. Course Outline.

ERIC Educational Resources Information Center

New York Inst. of Tech., Old Westbury.

This document contains the course syllabus and 12 independent practice modules for an introductory college algebra course that requires some previous knowledge of algebra and the ability to work at a rapid pace. Topics include the basic operations with signed integers; fractions; decimals; literal expressions; algebraic fractions; radicals;…

STUDYING ALBUMIN FRACTIONS OF THE BLOOD BY THE ELECTROPHORESIS METHOD OF CHRONIC MOUTH DISEASES,

DTIC Science & Technology

Conclusions: The examined patients showed an increase in globulin content and reduction in albumina . This displacement becomes more pronounced in... albumina fractions improved, histamine level decreased, general condition improved. This allows to continue with the perfection of basic therapy of the named illnesses. (Author)

Purification and fractionation of membranes for proteomic analyses.

PubMed

Marmagne, Anne; Salvi, Daniel; Rolland, Norbert; Ephritikhine, Geneviève; Joyard, Jacques; Barbier-Brygoo, Hélène

2006-01-01

Proteomics is a very powerful approach to link the information contained in sequenced genomes, such as Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. However, membrane proteomics remains a challenge. One way to bring into view the complex mixture of proteins present in a membrane is to develop proteomic analyses based on (1) the use of highly purified membrane fractions and (2) fractionation of membrane proteins to retrieve as many proteins as possible (from the most to the less hydrophobic ones). To illustrate such strategies, we choose two types of membranes, the plasma membrane and the chloroplast envelope membranes. Both types of membranes can be prepared in a reasonable degree of purity from different types of tissues: the plasma membrane from cultured cells and the chloroplast envelope membrane from whole plants. This article is restricted to the description of methods for the preparation of highly purified and characterized plant membrane fractions and the subsequent fractionation of these membrane proteins according to simple physicochemical criteria (i.e., chloroform/methanol extraction, alkaline or saline treatments) for further analyses using modern proteomic methodologies.

Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

PubMed Central

Alibolandi, Mona; Mirzahoseini, Hasan

2011-01-01

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. PMID:21837279

DOE Office of Scientific and Technical Information (OSTI.GOV)

Szcześ, Aleksandra, E-mail: aszczes@poczta.umcs.lublin.pl; Czemierska, Magdalena; Jarosz-Wilkołazka, Anna

Extracellular polymeric substance (EPS) extracted from Rhodococcus opacus bacterial strain was used as a matrix for calcium carbonate precipitation using the vapour diffusion method. The total exopolymer and water-soluble exopolymer fraction of different concentrations were spread on the mica surface by the spin-coating method. The obtained layers were characterized using the atomic force microscopy measurement and XPS analysis. The effects of polymer concentration, initial pH of calcium chloride solution and precipitation time on the obtained crystals properties were investigated. Raman spectroscopy and scanning electron microscopy were used to characterize the precipitated minerals. It was found that the type of precipitatedmore » CaCO{sub 3} polymorph and the crystal size depend on the kind of EPS fraction. The obtained results indicates that the water soluble fraction favours vaterite dissolution and calcite growth, whereas the total EPS stabilizes vaterite and this effect is stronger at basic pH. It seems to be due to different contents of the functional group of EPS fractions. - Highlights: • CaCO{sub 3} crystal size and polymorph can be controlled by EPS substance obtained from R. opacus. • The water soluble fraction favours vaterite dissolution and calcite growth. • The total EPS stabilizes vaterite. • This effect is stronger at basic pH.« less

[Elimination of toxic compounds, biological evaluation and partial characterization of the protein from jojoba meal (Simmondsia chinensis [Link] Schneider].

PubMed

Medina Juárez, L A; Trejo González, A

1989-12-01

The purpose of this study was to establish a new methodology to remove the toxic compounds present in jojoba meal and flour. Also, to perform the biological evaluation of the detoxified products and to chemically characterize the protein fractions. Jojoba meal and seed without testa were deffated with hexane and detoxified with a 7:3 isopropanol-water mixture which removed 86% of total phenolic compounds and 100% of simmondsins originally present, the resulting products had reduced bitterness and caused no deaths on experimental animals. NPR values obtained for diets containing such products were significantly different from those obtained with the casein control (p less than 0.05). Total protein was made up of three different fractions: the water-soluble fraction was the most abundant (61.8%), followed by the salt-soluble (23.6%), and the alkaline soluble fraction (14.6%). The nitrogen solubility curves showed that the isoelectric point for the water-soluble and salt-soluble fractions was pH 3.0, while that of the alkaline fraction fell in the range of 4.5-5.0. All fractions had a maximum solubility at pH 7.0. The methodology reported here, offers a viable solution to eliminate toxic compounds from jojoba meal or seeds, and upgrades the potential use of products such as animal feed or raw material for the production of protein isolates.

Modeling the evolution of protein domain architectures using maximum parsimony.

PubMed

Fong, Jessica H; Geer, Lewis Y; Panchenko, Anna R; Bryant, Stephen H

2007-02-09

Domains are basic evolutionary units of proteins and most proteins have more than one domain. Advances in domain modeling and collection are making it possible to annotate a large fraction of known protein sequences by a linear ordering of their domains, yielding their architecture. Protein domain architectures link evolutionarily related proteins and underscore their shared functions. Here, we attempt to better understand this association by identifying the evolutionary pathways by which extant architectures may have evolved. We propose a model of evolution in which architectures arise through rearrangements of inferred precursor architectures and acquisition of new domains. These pathways are ranked using a parsimony principle, whereby scenarios requiring the fewest number of independent recombination events, namely fission and fusion operations, are assumed to be more likely. Using a data set of domain architectures present in 159 proteomes that represent all three major branches of the tree of life allows us to estimate the history of over 85% of all architectures in the sequence database. We find that the distribution of rearrangement classes is robust with respect to alternative parsimony rules for inferring the presence of precursor architectures in ancestral species. Analyzing the most parsimonious pathways, we find 87% of architectures to gain complexity over time through simple changes, among which fusion events account for 5.6 times as many architectures as fission. Our results may be used to compute domain architecture similarities, for example, based on the number of historical recombination events separating them. Domain architecture "neighbors" identified in this way may lead to new insights about the evolution of protein function.

Modeling the Evolution of Protein Domain Architectures Using Maximum Parsimony

PubMed Central

Fong, Jessica H.; Geer, Lewis Y.; Panchenko, Anna R.; Bryant, Stephen H.

2007-01-01

Domains are basic evolutionary units of proteins and most proteins have more than one domain. Advances in domain modeling and collection are making it possible to annotate a large fraction of known protein sequences by a linear ordering of their domains, yielding their architecture. Protein domain architectures link evolutionarily related proteins and underscore their shared functions. Here, we attempt to better understand this association by identifying the evolutionary pathways by which extant architectures may have evolved. We propose a model of evolution in which architectures arise through rearrangements of inferred precursor architectures and acquisition of new domains. These pathways are ranked using a parsimony principle, whereby scenarios requiring the fewest number of independent recombination events, namely fission and fusion operations, are assumed to be more likely. Using a data set of domain architectures present in 159 proteomes that represent all three major branches of the tree of life allows us to estimate the history of over 85% of all architectures in the sequence database. We find that the distribution of rearrangement classes is robust with respect to alternative parsimony rules for inferring the presence of precursor architectures in ancestral species. Analyzing the most parsimonious pathways, we find 87% of architectures to gain complexity over time through simple changes, among which fusion events account for 5.6 times as many architectures as fission. Our results may be used to compute domain architecture similarities, for example, based on the number of historical recombination events separating them. Domain architecture “neighbors” identified in this way may lead to new insights about the evolution of protein function. PMID:17166515

Fractionation of Whey Protein Isolate with Supercritical Carbon Dioxide—Process Modeling and Cost Estimation

PubMed Central

Yver, Alexandra L.; Bonnaillie, Laetitia M.; Yee, Winnie; McAloon, Andrew; Tomasula, Peggy M.

2012-01-01

An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) from a commercial whey protein isolate (WPI) containing 20% α-LA and 55% β-LG, through selective precipitation of α-LA. Pilot-scale experiments were performed around the optimal parameter range (T = 60 to 65 °C, P = 8 to 31 MPa, C = 5 to 15% (w/w) WPI) to quantify the recovery rates of the individual proteins and the compositions of both fractions as a function of processing conditions. Mass balances were calculated in a process flow-sheet to design a large-scale, semi-continuous process model using SuperproDesigner® software. Total startup and production costs were estimated as a function of processing parameters, product yield and purity. Temperature, T, pressure, P, and concentration, C, showed conflicting effects on equipment costs and the individual precipitation rates of the two proteins, affecting the quantity, quality, and production cost of the fractions considerably. The highest α-LA purity, 61%, with 80% α-LA recovery in the solid fraction, was obtained at T = 60 °C, C = 5% WPI, P = 8.3 MPa, with a production cost of $8.65 per kilogram of WPI treated. The most profitable conditions resulted in 57%-pure α-LA, with 71% α-LA recovery in the solid fraction and 89% β-LG recovery in the soluble fraction, and production cost of $5.43 per kilogram of WPI treated at T = 62 °C, C = 10% WPI and P = 5.5 MPa. The two fractions are ready-to-use, new food ingredients with a pH of 6.7 and contain no residual acid or chemical contaminants. PMID:22312250

Cholesterol-lowering effect of kori-tofu protein and its high-molecular-weight fraction content.

PubMed

Ishiguro, Takahiro; Tatsunokuchi, Seiji; Mitsui, Nobuo; Kayahara, Hisataka; Murasawa, Hisashi; Konishi, Yotaro; Nagaoka, Satoshi

2011-01-01

The serum total cholesterol concentration was significantly lower in the kori-tofu feeding group than in the soy protein isolate (SPI) group, except on the 28th day of the experiment. The high-molecular-weight fraction (HMF) content of the kori-tofu protein was significantly higher than that of SPI. This difference in the HMF content may have influenced the cholesterol-lowering effect of the protein.

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

Comprehensive Evaluation of Anti-hyperglycemic Activity of Fractionated Momordica charantia Seed Extract in Alloxan-Induced Diabetic Rats

PubMed Central

Choudhary, Shailesh Kumar; Chhabra, Gagan; Sharma, Dipali; Vashishta, Aruna; Ohri, Sujata; Dixit, Aparna

2012-01-01

The present study evaluates anti-hyperglycemic activity of fractionated Momordica charantia (bitter gourd) seed extracts. Fasting blood glucose levels were evaluated before and after administration of different fractions of the seed extract. Among the three fractions tested, fraction Mc-3 (15 mg/kg b.wt.) showed the maximum anti-hyperglycemic activity and reduced blood glucose levels in experimental diabetic rats significantly. The activities of the key regulatory enzymes of glucose metabolism (hexokinase, pyruvate kinase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase) were determined in Mc-3-treated diabetic animals. Once-daily administration of the fraction Mc-3 for prolonged period of 18 days to the experimental diabetic animals did not result in any nephrotoxicity or hepatotoxicity as evident from insignificant changes in biochemical parameters indicative of liver and kidney functions. Further fractionation of the fraction Mc-3 by size exclusion chromatography resulted in a fraction, designated Mc-3.2, possessing anti-hyperglycemic activity. The fraction Mc-3.2 showed the presence of a predominant protein band of ~11 kDa on SDS-PAGE. Loss in anti-hyperglycemic activity of the Mc-3.2 upon protease treatment indicates the proteinaceous nature of the anti-hyperglycemic principles. Overall, the results suggest that Momordica charantia seeds contain an effective anti-hyperglycemic protein(s) which may find application in treatment of diabetes without evident toxic effects. PMID:23320026

Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry

PubMed Central

Griaud, François; Denefeld, Blandine; Lang, Manuel; Hensinger, Héloïse; Haberl, Peter; Berg, Matthias

2017-01-01

ABSTRACT Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner. PMID:28379786

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

PubMed

Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

2017-06-02

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

Refractory periorbital edema in a 29-year-old man.

PubMed

Li, J T; Garrity, J A; Kephart, G M; Gleich, G J

1992-08-01

A 29-year-old man developed periorbital edema which was initially diagnosed as angioedema. Further clinical investigation by orbital CT and orbital biopsy showed this to be orbital pseudotumor. Immunofluorescence staining for major basic protein clearly demonstrated tissue eosinophilia and extracellular major basic protein deposition. Orbital pseudotumor can mimic angioedema or allergic rhinoconjunctivitis and should be familiar to allergists and primary care physicians. The demonstration of striking extracellular major basic protein in biopsy specimen implicates a role for eosinophils in the pathogenesis of this disease.

Using Every Pupil Response in Mathematics Instruction.

ERIC Educational Resources Information Center

Lauritzen, Carol

1985-01-01

Discusses the "Every Pupil Response" (EPR) strategy and its use in teaching basic facts, problem-solving, place value, and fractions. Basically, the technique involves children responding simultaneously to a question by holding up a card, using parts of their bodies, or stick figures. Advantages of EPR are noted. (JN)

Basic Applied Mathematics Part 1.

ERIC Educational Resources Information Center

New York City Board of Education, Brooklyn, NY. Div. of Curriculum and Instruction.

This guide, published by the New York City Board of Education, presents 62 lesson plans in basic mathematics for tenth grade students. Lesson plans and performance objectives focus on the following areas: (1) fundamental operations with signed numbers; (2) linear, weight and temperature measurements; (3) fractions, decimals and percents; (4)…

S.M.P. SEQUENTIAL MATHEMATICS PROGRAM.

ERIC Educational Resources Information Center

CICIARELLI, V; LEONARD, JOSEPH

A SEQUENTIAL MATHEMATICS PROGRAM BEGINNING WITH THE BASIC FUNDAMENTALS ON THE FOURTH GRADE LEVEL IS PRESENTED. INCLUDED ARE AN UNDERSTANDING OF OUR NUMBER SYSTEM, AND THE BASIC OPERATIONS OF WORKING WITH WHOLE NUMBERS--ADDITION, SUBTRACTION, MULTIPLICATION, AND DIVISION. COMMON FRACTIONS ARE TAUGHT IN THE FIFTH, SIXTH, AND SEVENTH GRADES. A…

Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

1988-08-01

ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost,more » a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.« less

The fractional dynamics of quantum systems

NASA Astrophysics Data System (ADS)

Lu, Longzhao; Yu, Xiangyang

2018-05-01

The fractional dynamic process of a quantum system is a novel and complicated problem. The establishment of a fractional dynamic model is a significant attempt that is expected to reveal the mechanism of fractional quantum system. In this paper, a generalized time fractional Schrödinger equation is proposed. To study the fractional dynamics of quantum systems, we take the two-level system as an example and derive the time fractional equations of motion. The basic properties of the system are investigated by solving this set of equations in the absence of light field analytically. Then, when the system is subject to the light field, the equations are solved numerically. It shows that the two-level system described by the time fractional Schrödinger equation we proposed is a confirmable system.

The impact of newly produced protein and dietary fiber rich fractions of yellow pea (Pisum sativum L.) on the structure and mechanical properties of pasta-like sheets.

PubMed

Muneer, Faraz; Johansson, Eva; Hedenqvist, Mikael S; Plivelic, Tomás S; Markedal, Keld Ejdrup; Petersen, Iben Lykke; Sørensen, Jens Christian; Kuktaite, Ramune

2018-04-01

Two fractions from pea (Pisum sativum L.), protein isolate (PPI) and dietary fiber (PF), were newly produced by extraction-fractionation method and characterized in terms of particle size distribution and structural morphology using SEM. The newly produced PPI and PF fractions were processed into pasta-like sheets with varying protein to fiber ratios (100/0, 90/10, 80/20, 70/30 and 50/50, respectively) using high temperature compression molding. We studied protein polymerization, molecular structure and protein-fiber interactions, as well as mechanical performance and cooking characteristics of processed PPI-PF blends. Bi-modal particle size distribution and chemical composition of the PPI and PF fractions influenced significantly the physicochemical properties of the pasta-like sheets. Polymerization was most pronounced for the 100 PPI, 90/10 and 80/20 PPI-PF samples as studied by SE-HPLC, and polymerization decreased with addition of the PF fraction. The mechanical properties, as strength and extensibility, were likewise the highest for the 100 PPI and 90/10 PPI-PF blends, while the E-modulus was similar for all the studied blends (around 38 MPa). The extensibility decreased with the increasing amount of PF in the blend. The highest amounts of β-sheets were found in the pasta-like sheets with high amounts of PPI (100, 90 and 80%), by FT-IR. An increase in PF fraction in the blend, resulted into the high amounts of unordered structures as observed by FT-IR, as well as in an increase in the molecular scattering distances observed by SAXS. The water uptake increased and cooking loss decreased with increased proportions of the PF fraction, and the consistency of 10 min cooked pasta-like sheets were alike al dente texture. The new knowledge obtained in this study on the use of extraction-fractionation method to produce novel PPI and PF fractions for developing innovative high nutritious food can be of a great importance. The obtained knowledge on the pea protein and fiber processing behaviour could greatly contribute to a better control of functional properties of various temperature-processed products from yellow pea. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C and mitogenic factor MF.

PubMed Central

Schmidt, K H; Gerlach, D; Wollweber, L; Reichardt, W; Mann, K; Ozegowski, J H; Fleischer, B

1995-01-01

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein. PMID:7591107

[Heterogeneity of brain-specific proteins of S100 type].

PubMed

Poletaev, A B; Kupriianenko, T I

1980-12-01

A procedure for preparative separation of proteins from bovine brain precipitated in a saturated solution by ammonium sulfate (pH 4.0) and remaining in the supernatant is described. It was shown that not less than 22 fractions (up to 28 fractions considering the "equivocal" reaction) produce different responses to the antiserum against the major protein, i.e. S100.

Binding of [35S]saccharin to a protein fraction of rat tongue epithelia.

PubMed

Shimazaki, K; Sato, M; Takegami, T

1981-11-05

The binding of [35S]saccharin to ammonium sulfate fractions from homogenates of rat tongue epithelia was measured by equilibrium dialysis. The 40--60% saturated ammonium sulfate fraction from the buffer-soluble fraction had the highest saccharin-binding activity. Binding of [35S]saccharin to the 40--60% ammonium sulfate fraction was inhibited by unlabeled saccharin sodium salt. The inhibition increased with increasing unlabeled saccharin concentration and was nearly complete above 10 mM. [35S]Saccharin binding to the 40--60% ammonium sulfate fraction extracted from the tongue epithelia was inhibited by glucose, lactose and sucrose, while binding to similar fractions from tongue muscle was not affected by these sugars. The inhibition of binding of labeled saccharin to the epithelial fraction increased with increasing glucose concentrations. About 35% of the binding was inhibited by 1 M glucose. No significant difference in the amount of inhibition was seen among the three sugars at 0.1 M. The 40--60% ammonium sulfate fraction from tongue epithelium devoid of taste buds bound much less [35S]saccharin than did a similar fraction from epithelium with taste buds. Binding of [35S]saccharin by the preparation from epithelium devoid of taste buds was not inhibited by glucose. The results provide evidence that the 40--60% ammonium sulfate fraction from tongue epithelia with taste buds contains a protein which binds saccharin and sugars. We hypothesize that it is a sweet taste receptor protein.

Mutagenicity of basic fractions derived from lamb and beef cooked by common household methods.

PubMed

Barrington, P J; Baker, R S; Truswell, A S; Bonin, A M; Ryan, A J; Paulin, A P

1990-03-01

Mutagen production was examined in lamb and beef in relation to certain common household cooking methods. Mutagenicity was assessed, after extraction of the basic fraction of cooked meat samples, using Salmonella typhimurium strain TA1538 with added rat-liver S-9 homogenate. Little or no mutagenicity was found in barbecued lamb chops, in microwave-cooked lamb chops, sirloin steak, leg of lamb, or rolled beef loaf, in roasted leg of lamb or rolled beef loaf, in stewed blade steak or in boiled chuck steak. However, the basic fraction from well-done, edible fried or grilled meat contained mutagenic activity equivalent to approximately 30,000 TA1538 revertants/100 g cooked meat. It was found tht the mutagenic activity of grilled lamb chops, sirloin and rump steaks was directly related to the average surface temperatures attained during cooking. Use of butter as a frying medium was particularly associated with higher mutagenicity in meat samples. Fried meats (rump and fillet steaks) generally yielded higher mutagenic activity than did grilled meats (rump steak, lamb chops) at comparable temperatures of the cooking medium. Using similar cooking procedures, lamb did not differ markedly from beef in mutagenic activity.

Proteins in Relation to Vigor and Viability of White Lupin (Lupinus albus L.) Seed Stored for 26 Years

PubMed Central

Dobiesz, Malwina; Piotrowicz-Cieślak, Agnieszka I.

2017-01-01

The aim of the study was to evaluate the vigor and viability as well as to determine and compare the contents of selected protein fractions of white lupin (Lupinus albus L.) seeds stored for 26 years at temperatures of -14°C and +20°C. The seeds stored at -14°C germinated in 86.3%, while the seeds stored at +20°C did not germinate at all. The viability evaluation was confirmed by the measuring electroconductivity of seed exudates. In seeds stored at -14°C the contents of γ, δ, and β conglutin were 14, 4 and 69 mg g-1 fresh mass, respectively, while in seed stored at +20°C they were 15.5, 3, 65 mg g-1 fresh mass, respectively. One-dimensional electrophoresis of γ and δ conglutin fractions indicated the presence of several intense polypeptide bands with molecular weights from 23.0 to 10.3 kDa. Polypeptide bands with a molecular weight of 22.4 and 19.8 kDa exhibited almost two times higher expression in the seeds stored at -14°C compared to the seeds stored at +20°C. Electrophoresis revealed 310 protein spots on the maps generated for seeds stored at -14°C, and 228 spots for seeds stored at +20°C. In seeds stored at +20°C most polypeptide subunits had a pI ranging from 4.5 to 7 and a molecular weight of 10–97 kDa. The greatest differences in the contents of polypeptides between the analyzed variants was observed within the range of 20–45 kDa (-14°C: 175, +20°C: 115 protein spots) and within the range of 65–97 kDa (-14°C: 103, +20°C: 75 protein spots). In seeds stored at +20°C, a clear decline in basic (8–10 pI) polypeptides was observed. The study demonstrated that the polypeptides identified as γ and δ conglutins are probably closely related to vigor and viability of seeds. PMID:28848591

ISOLATION AND CHARACTERIZATION OF LAMELLAR BODIES AND TUBULAR MYELIN FROM RAT LUNG HOMOGENATES

PubMed Central

Gil, Joan; Reiss, Oscar K.

1973-01-01

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins. PMID:4726305

Protein identification and quantification from riverbank grape, Vitis riparia: Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic analysis.

PubMed

George, Iniga S; Fennell, Anne Y; Haynes, Paul A

2015-09-01

Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in-gel digestion and in-solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS-PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP-GPF was used. FASP-GPF is more reproducible, less expensive and a better method than SDS-PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 (http://proteomecentral.proteomexchange.org/dataset/PXD001399). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.

PubMed

Wang, Michael Z; Howard, Brandon; Campa, Michael J; Patz, Edward F; Fitzgerald, Michael C

2003-09-01

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.

Role of linguistic skills in fifth-grade mathematics.

PubMed

Kleemans, Tijs; Segers, Eliane; Verhoeven, Ludo

2018-03-01

The current study investigated the direct and indirect relations between basic linguistic skills (i.e., phonological skills and grammatical ability) and advanced linguistic skills (i.e., academic vocabulary and verbal reasoning), on the one hand, and fifth-grade mathematics (i.e., arithmetic, geometry, and fractions), on the other, taking working memory and general intelligence into account and controlling for socioeconomic status, age, and gender. The results showed the basic linguistic representations of 167 fifth graders to be indirectly related to their geometric and fraction skills via arithmetic. Furthermore, advanced linguistic skills were found to be directly related to geometry and fractions after controlling for arithmetic. It can be concluded that linguistic skills directly and indirectly relate to mathematical ability in the upper grades of primary education, which highlights the importance of paying attention to such skills in the school curriculum. Copyright © 2017 Elsevier Inc. All rights reserved.

Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semelr, B.L.; Anderson, C.W.; Hanecak, R.

1982-02-01

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNAmore » sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.« less

Concentrated Solutions of Single-Chain Nanoparticles: A Simple Model for Intrinsically Disordered Proteins under Crowding Conditions.

PubMed

Moreno, Angel J; Lo Verso, Federica; Arbe, Arantxa; Pomposo, José A; Colmenero, Juan

2016-03-03

By means of large-scale computer simulations and small-angle neutron scattering (SANS), we investigate solutions of single-chain nanoparticles (SCNPs), covering the whole concentration range from infinite dilution to melt density. The analysis of the conformational properties of the SCNPs reveals that these synthetic nano-objects share basic ingredients with intrinsically disordered proteins (IDPs), as topological polydispersity, generally sparse conformations, and locally compact domains. We investigate the role of the architecture of the SCNPs in their collapse behavior under macromolecular crowding. Unlike in the case of linear macromolecules, which experience the usual transition from self-avoiding to Gaussian random-walk conformations, crowding leads to collapsed conformations of SCNPs resembling those of crumpled globules. This behavior is already found at volume fractions (about 30%) that are characteristic of crowding in cellular environments. The simulation results are confirmed by the SANS experiments. Our results for SCNPs--a model system free of specific interactions--propose a general scenario for the effect of steric crowding on IDPs: collapse from sparse conformations at high dilution to crumpled globular conformations in cell environments.

Activity of maize transglutaminase overexpressed in Escherichia coli inclusion bodies: an alternative to protein refolding.

PubMed

Carvajal, Patricia; Gibert, Jordi; Campos, Nefertiti; Lopera, Oriol; Barberà, Eduard; Torné, Jose M; Santos, Mireya

2011-01-01

Transglutaminases (TGases) catalyze protein post-translational modification by ε-(γ-glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS-PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI-TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large-scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant-substrate preference. Solubilization of the IB fraction with Triton X-100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Metalloproteomics Approach to Analyze Mercury in Breast Milk and Hair Samples of Lactating Women in Communities of the Amazon Basin, Brazil.

PubMed

Cerbino, M R; Vieira, José Cavalcante Souza; Braga, C P; Oliveira, G; Padilha, I F; Silva, T M; Zara, L F; Silva, N J; Padilha, P M

2018-02-01

Mercury is a potentially toxic element that is present in the environment of the Brazilian Amazon and is responsible for adverse health effects in humans. This study sought to assess possible protein biomarkers of mercury exposure in breast milk samples from lactating women in the Madeira and Negro Rivers in the Brazilian Amazon. The mercury content of hair samples of lactating women was determined, and the proteome of breast milk samples was obtained using two-dimensional electrophoresis after protein precipitation with acetone. Mercury measurements of protein spots obtained via protein fractionation were performed by graphite furnace atomic absorption spectrometry (GFAAS), and it was observed that mercury is linked to proteins with molecular masses in the range of 14-26 kDa. The total mercury concentration was also determined by GFAAS in unprocessed milk, lyophilized milk, and protein pellets, with the purpose of determining the mercury mass balance in relation to the concentration of this element in milk and pellets. Approximately 85 to 97% of mercury present in the lyophilized milk from samples of lactating women of the Madeira River is bound in the protein fraction. From lactating women of the Negro River, approximately 49% of the total mercury is bound in the protein fraction, and a difference of 51% is bound in the lipid fraction.

A streamlined isolation method and the autoxidation profiles of tuna myoglobin.

PubMed

Nurilmala, Mala; Ushio, Hideki; Watabe, Shugo; Ochiai, Yoshihiro

2018-05-01

Determination of the redox state of myoglobin (Mb) gives useful information for evaluating the quality of tuna meat. To attain this purpose, a fast streamlined method has been established basically based on preparative native gel electrophoresis to isolate Mb from the dark muscle of Pacific bluefin tuna. Crude Mb fraction was prepared from dark muscle by ammonium sulfate saturation fractionation and subsequently Mb was purified by preparative native gel electrophoresis under the isoelectric pH of the Mb, resulting in absorption (or trapping) of all the contaminating proteins in the gel. Purified Mb was converted to oxy form with a trace amount of sodium hydrosulfite, and subsequently dialyzed against 50 mM sodium citrate (pH 5.6) or 50 mM sodium phosphate (pH 6.5). The purified tuna Mb was examined for the temperature and pH dependencies of autoxidation using horse Mb as a reference. Tuna Mb was oxidized 2.5-3 times faster than horse Mb irrespective of the pH conditions examined. The highest autoxidation rates both at 0 and 37 °C were observed at pH 5.6. These data were comparable to those obtained for Mbs isolated by conventional chromatographic methods.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

PubMed

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

The kin17 Protein in Murine Melanoma Cells

PubMed Central

Ramos, Anelise C.; Gaspar, Vanessa P.; Kelmer, Sabrina M. G.; Sellani, Tarciso A.; Batista, Ana G. U.; De Lima Neto, Quirino A.; Rodrigues, Elaine G.; Fernandez, Maria A.

2015-01-01

kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma. PMID:26610484

Oily fraction of Semecarpus anacardium Linn nuts involves protein kinase C activation for its pro-inflammatory response.

PubMed

Tripathi, Yamini B; Pandey, Nidhi; Tripathi, Deepshikha; Tripathi, Pratibha

2010-12-01

The oily fraction (non polar fraction-NPF) of S. anacardium (SA) significantly increased the expression of protein kinase C-delta (PKC-delta) in macrophages in concentration dependent manner, which was similar to phorbol myristate acetate (PMA) response. Further, H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine), an inhibitor of PKC significantly inhibited this NPF mediated response in a concentration dependent manner. In the post treatment kinetics, H-7 showed this inhibition only up to 6 min post NPF/PMA addition, but in similar condition, quercetin, a flavone with reported antioxidant property, showed this inhibition only up to 2 min. The results clearly suggest that oily fraction of SA nuts enhances the expression of PKC protein, which may be responsible for its reported pro-inflammatory property.

Composition and immunoreactivity of the A60 complex and other cell fractions from Mycobacterium bovis BCG.

PubMed

Cocito, C; Vanlinden, F

1995-02-01

Surface static cultures of Mycobacterium bovis BCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 6O (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29-45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS-PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60- and non-A60-proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS-PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.

Proteomic analysis of duck fatty liver during post-mortem storage related to the variability of fat loss during cooking of "foie gras".

PubMed

Theron, Laetitia; Fernandez, Xavier; Marty-Gasset, Nathalie; Chambon, Christophe; Viala, Didier; Pichereaux, Carole; Rossignol, Michel; Astruc, Thierry; Molette, Caroline

2013-01-30

Fat loss during cooking of duck "foie gras" is the main problem for both manufacturers and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yields remains high and uncontrolled. To understand the biochemical effects of postslaughter processing on fat loss during cooking, this study characterizes for the first time the protein expression of fatty liver during chilling using a proteomic approach. For this purpose the proteins were separated according to their solubility: the protein fraction soluble in a buffer of low ionic strength (S) and the protein fraction insoluble in the same buffer (IS). Two-dimensional electrophoresis was used to analyze the S fraction and mass spectrometry for the identification of spots of interest. This analysis revealed 36 (21 identified proteins) and 34 (26 identified proteins) spots of interests in the low-fat-loss and high-fat-loss groups, respectively. The expression of proteins was lower after chilling, which revealed a suppressive effect of chilling on biological processes. The shot-gun strategy was used to analyze the IS fraction, with the identification of all the proteins by mass spectrometry. This allowed identification of 554 and 562 proteins in the low-fat-loss and high-fat-loss groups, respectively. Among these proteins, only the proteins that were up-regulated in the high-fat-loss group were significant (p value = 3.17 × 10(-3)) and corresponded to protein from the cytoskeleton and its associated proteins. Taken together, these results suggest that the variability of technological yield observed in processing plants could be explained by different aging states of fatty livers during chilling, most likely associated with different proteolytic patterns.

Isolation of basal membrane proteins from BeWo cells and their expression in placentas from fetal growth-restricted pregnancies.

PubMed

Oh, Soo-Young; Hwang, Jae Ryoung; Lee, Yoonna; Choi, Suk-Joo; Kim, Jung-Sun; Kim, Jong-Hwa; Sadovsky, Yoel; Roh, Cheong-Rae

2016-03-01

The syncytiotrophoblast, a key barrier between the mother and fetus, is a polarized epithelium composed of a microvillus and basal membrane (BM). We sought to characterize BM proteins of BeWo cells in relation to hypoxia and to investigate their expression in placentas from pregnancies complicated by fetal growth restriction (FGR). We isolated the BM fraction of BeWo cells by the cationic colloidal silica method and identified proteins enriched in this fraction by mass spectrometry. We evaluated the effect of hypoxia on the expression and intracellular localization of identified proteins and compared their expression in BM fractions of FGR placentas to those from normal pregnancies. We identified BM proteins from BeWo cells. Among BM proteins, we further characterized heme oxygenase-1 (HO-1), voltage-dependent anion channel-1 (VDAC1), and ribophorin II (RPN2), based on their relevance to placental biology. Hypoxia enhanced the localization of these proteins to the BM of BeWo cells. HO-1, VDAC1, and RPN2 were selectively expressed in the human placental BM fraction. C-terminally truncated HO-1 was identified in placental BM fractions, and its BM expression was significantly reduced in FGR placentas than in normal placentas. Interestingly, a truncated HO-1 construct was predominantly localized in the BM in response to hypoxia and co-localized with VDAC1 in BeWo cells. Hypoxia increased the BM localization of HO-1, VDAC1, and RPN2 proteins. FGR significantly reduced the expression of truncated HO-1, which was surmised to co-localize with VDAC1 in hypoxic BeWo cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Consolidation of proteomics data in the Cancer Proteomics database.

PubMed

Arntzen, Magnus Ø; Boddie, Paul; Frick, Rahel; Koehler, Christian J; Thiede, Bernd

2015-11-01

Cancer is a class of diseases characterized by abnormal cell growth and one of the major reasons for human deaths. Proteins are involved in the molecular mechanisms leading to cancer, furthermore they are affected by anti-cancer drugs, and protein biomarkers can be used to diagnose certain cancer types. Therefore, it is important to explore the proteomics background of cancer. In this report, we developed the Cancer Proteomics database to re-interrogate published proteome studies investigating cancer. The database is divided in three sections related to cancer processes, cancer types, and anti-cancer drugs. Currently, the Cancer Proteomics database contains 9778 entries of 4118 proteins extracted from 143 scientific articles covering all three sections: cell death (cancer process), prostate cancer (cancer type) and platinum-based anti-cancer drugs including carboplatin, cisplatin, and oxaliplatin (anti-cancer drugs). The detailed information extracted from the literature includes basic information about the articles (e.g., PubMed ID, authors, journal name, publication year), information about the samples (type, study/reference, prognosis factor), and the proteomics workflow (Subcellular fractionation, protein, and peptide separation, mass spectrometry, quantification). Useful annotations such as hyperlinks to UniProt and PubMed were included. In addition, many filtering options were established as well as export functions. The database is freely available at http://cancerproteomics.uio.no. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular Weight Affected Antioxidant, Hypoglycemic and Hypotensive Activities of Cold Water Extract from Pleurotus citrinopileatus.

PubMed

Chen, Pao-Huei; Weng, Yih-Ming; Lin, Shu-Mei; Yu, Zer-Ran; Wang, Be-Jen

2017-10-01

Cold water extract of P. citrinopileatus (CWEPC) was fractioned into 4 fractions, PC-I (<1 kDa), PC-II (1-3.5 kDa), PC-III (3.5-10 kDa), and PC-IV (>10 kDa), by ultrafiltration. The antioxidant activities, the inhibition of pancreatic α-amylase, intestinal α-glucosidase, and hypertension-linked angiotensin converting enzyme (ACE), as well as the contents of polysaccharides, protein, and phenolic compounds of 4 fractions were determined. The results showed that lower MW fractions exerted a higher antioxidant activity, which was correlated to phenolic contents. The high molecular fraction (PC-IV) exhibited significantly higher inhibitory activity on α-amylase, α-glucosidase, and ACE compared to CWEPC and the other 3 lower MW fractions (<10 kDa), which was more related to protein contents. The inhibition capability of CWEPC and PC-IV on α-amylase activity was 1/13.4 to 1/2.7 relative to that of acarbose, respectively. Kinetic data revealed that PC-IV fraction followed a noncompetitive inhibition pattern on α-glucosidase activity. The study demonstrated that various MW fractions and types of components contribute to different biological functions of P. citrinopileatus and it is protein constituents but not peptides responsible for the hypoglycemic potential of CWEPC. © 2017 Institute of Food Technologists®.

Cocoa procyanidins and human cytokine transcription and secretion.

PubMed

Mao, T; Van De Water, J; Keen, C L; Schmitz, H H; Gershwin, M E

2000-08-01

We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

Structure of cortical cytoskeleton in fibers of mouse muscle cells after being exposed to a 30-day space flight on board the BION-M1 biosatellite.

PubMed

Ogneva, I V; Maximova, M V; Larina, I M

2014-05-15

The aim of the work was to analyze changes in the organization of the cortical cytoskeleton in fibers of the mouse soleus muscle, tibialis anterior muscle and left ventricular cardiomyocytes after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). The transversal stiffness of the cortical cytoskeleton of the cardiomyocytes and fibers of the skeletal muscles did not differ significantly within the study groups compared with the vivarium control group. The content of beta- and gamma-actin in the membranous fraction of proteins in the left ventricular cardiomyocytes did not differ significantly within all study groups and correlated with the transversal stiffness. A similar situation was revealed in fibers of the soleus muscle and tibialis anterior muscle. At the same time, the content of beta-actin in the cytoplasmic fraction of proteins was found to be decreased in all types of studied tissues compared with the control levels in the postflight group, with lowered beta-actin gene expression rates in the postflight group. After completion of the space flight, the content of alpha-actinin-4 was found to be reduced in the membranous fraction of proteins from the mouse cardiomyocytes, while its content in the cytoplasmic fraction of proteins did not change significantly. Furthermore, gene expression rates of this protein were decreased at the time of dissection (it was started after 13 h after landing). At the same time, the content of alpha-actinin-1 decreased in the membranous fraction and increased in the cytoplasmic fraction of proteins from the soleus muscle fibers. Copyright © 2014 the American Physiological Society.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

PubMed

Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

2017-08-01

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

Positively charged mini-protein Zbasic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports.

PubMed

Bolivar, Juan M; Nidetzky, Bernd

2012-07-03

Silica is a highly attractive support material for protein immobilization in a wide range of biotechnological and biomedical-analytical applications. Without suitable derivatization, however, the silica surface is not generally usable for attachment of proteins. We show here that Z(basic2) (a three α-helix bundle mini-protein of 7 kDa size that exposes clustered positive charges from multiple arginine residues on one side) functions as highly efficient silica binding module (SBM), allowing chimeras of target protein with SBM to become very tightly attached to underivatized glass at physiological pH conditions. We used two enzymes, d-amino acid oxidase and sucrose phosphorylase, to demonstrate direct immobilization of Z(basic2) protein from complex biological samples with extremely high selectivity. Immobilized enzymes displayed full biological activity, suggesting that their binding to the glass surface had occurred in a preferred orientation via the SBM. We also show that charge complementarity was the main principle of affinity between SBM and glass surface, and Z(basic2) proteins were bound in a very strong, yet fully reversible manner, presumably through multipoint noncovalent interactions. Z(basic2) proteins were immobilized on porous glass in a loading of 30 mg protein/g support or higher, showing that attachment via the SBM combines excellent binding selectivity with a technically useful binding capacity. Therefore, Z(basic2) and silica constitute a fully orthogonal pair of binding module and insoluble support for oriented protein immobilization, and this opens up new opportunities for the application of silica-based materials in the development of supported heterogeneous biocatalysts.

Rheological, physico-sensory, nutritional and storage characteristics of bread enriched with roller milled fractions of black gram (Phaseolus mungo L.).

PubMed

Indrani, D; Sakhare, Suresh D; Milind; Inamdar, Aashitosh A

2015-08-01

Black gram grains were fractionated using roller flour mill. Effect of combination of additives (CA) namely dry gluten powder, sodium stearoyl-2-lactylate, fungal α-amylase on the rheological and bread making characteristics of wheat flour partly replaced with roller milled fractions of black gram was studied. With increase in the addition of straight run flour (SRF), protein rich fraction (PRF), protein and fiber rich fraction (P&FRF) from 0 to 20 %, fiber rich fraction, FRF (0-15 %), the farinograph water absorption increased and dough stability decreased; amylograph pasting temperature increased and peak viscosity decreased; bread volume decreased and crumb firmness value increased indicating adverse effect of these fractions on the rheological and bread making characteristics of wheat flour. Sensory evaluation showed that breads were acceptable only up to the level of 15 % for SRF, PRF & P&FRF and 10 % for FRF. However, when CA containing dry gluten powder, sodium stearoyl-2-lactylate and fungal α-amylase was incorporated the overall quality of the products improved. Use of these fractions increased the protein and fiber contents of bread by 1.24-1.66 and 1.48-3.79 times respectively. The results showed that possibility of utilising roller milled black gram fractions along with CA to improve the taste, texture and nutritional quality of bread.

Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers.

PubMed

Teulé, Florence; Addison, Bennett; Cooper, Alyssa R; Ayon, Joel; Henning, Robert W; Benmore, Chris J; Holland, Gregory P; Yarger, Jeffery L; Lewis, Randolph V

2012-06-01

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline-containing pentapeptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = Y/S or A/A, respectively. Post-fiber processing involved similar drawing ratios (2-2.5×) before or after water-treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. Nuclear magnetic resonance indicated that the fraction of β-sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water-treatment. X-ray diffraction showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three-dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk-like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif re-design impacted protein self-assembly mechanisms and requirements for fiber formation. Copyright © 2011 Wiley Periodicals, Inc.

Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers

PubMed Central

Teulé, Florence; Addison, Bennett; Cooper, Alyssa R.; Ayon, Joel; Henning, Robert W.; Benmore, Chris J.; Holland, Gregory P.; Yarger, Jeffery L.; Lewis, Randolph V.

2012-01-01

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline-containing pentaptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX1 GPGGX2)2 with X1/X2=A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX1 GPGGX2)2 with X1/X2 = Y/S or A/A, respectively. Post fiber processing involved similar drawing ratios (2–2.5×) before or after water-treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. NMR indicated that the fraction of β-sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water-treatment. XRD showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three-dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk-like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif redesign impacted protein self-assembly mechanisms and requirements for fiber formation. PMID:22012252

Antinutritional factors and functionality of protein-rich fractions of industrial guar meal as affected by heat processing.

PubMed

Nidhina, N; Muthukumar, S P

2015-04-15

Proximate composition analysis and antinutritional factor composition of different fractions of industrial guar meal: raw churi (IRC), heated churi (IHC), final churi (IFC) and guar korma (IGK) were studied and compared. Protein content was found to be very high in IGK (52.7%) when compared to the churi fractions (32-33%) and the trypsin inhibitor activities were found to be negligible in all the fractions (0.58-1.8 mg/g). Single fraction (IGK) was selected for further studies, based on the protein content. The antinutritional factors of selected fractions were significantly reduced by different heat treatments. Heat treatments significantly increased the water absorbing capacity of IGK, but reduced the nitrogen solubility, emulsifying and foaming capacity. Highest L(∗) value was observed for boiled IGK, highest a(∗) and b(∗) values for roasted IGK, during colour measurement. FTIR spectral analysis revealed the presence several aromatic groups in IGK and slight modifications in the molecular structure during heat treatments. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inhibition of protein glycation by procyanidin-B2 enriched fraction of cinnamon: delay of diabetic cataract in rats.

PubMed

Muthenna, Puppala; Raghu, Ganugula; Akileshwari, Chandrasekhar; Sinha, Sukesh Narayana; Suryanarayana, Palla; Reddy, Geereddy Bhanuprakash

2013-11-01

Accumulation of advanced glycation endproducts (AGE) from nonenzymatic glycation of proteins has been implicated in several diabetic complications including diabetic cataract. Previously, we have reported that extracts of dietary agents such as cinnamon have the potential to inhibit AGE formation. In this study, we have shown procyanidin-B2 as the active component of cinnamon that is involved in AGE inhibition using bioassay-guided fractionation of eye lens proteins under in vitro conditions. The data indicate that procyanidin-B2 enriched fraction scavenges dicarbonyls. Further, procyanidin-B2 fraction of cinnamon inhibited the formation of glycosylated hemoglobin in human blood under ex vivo conditions. We have also demonstrated the physiological significance of procyanidin-B2 fraction in terms of delay of diabetic cataract through inhibition of AGE in diabetic rats. These findings establish the antiglycating potential of procyanidin-B2 fraction of cinnamon which suggests a scope for controlling AGE-mediated diabetic complications by food sources that are rich in proanthocyanidins like procyanidin-B2. © 2013 International Union of Biochemistry and Molecular Biology.

Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

NASA Technical Reports Server (NTRS)

Schneegurt, M. A.; Sherman, D. M.; Sherman, L. A.; Mitchell, C. A. (Principal Investigator)

1997-01-01

Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

PubMed

Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

2018-02-20

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

The effect of hypodynamia on mineral and protein metabolism in calcified tissues of the maxillodental system (experimental radioisotope study)

NASA Technical Reports Server (NTRS)

Prokhonchukov, A. A.; Kovalenko, Y. A.; Kolesnik, A. G.; Kondratyev, Y. I.; Ilyushko, N. A.

1980-01-01

Mineral and protein metabolism was studied in experiments on 60 white rats, using P-32 and Ca-45 uptake in the mineral fractions, 2C-14-glycine in the protein fractions, and P-32 in both fractions of calcified tissues as indices over a 100 day period of experimental hypodynamia. Combined alterations in mineral and protein metabolism occurred in the calcified tissues of the experimental animals. The most pronounced changes were found in P-32 and 2C-14-glycine metabolism. In the incisors and femoral bones, these alterations occurred in two phases: P-32 and 2C-14-glycine uptake first increased, then decreased. Changes in Ca-45 metabolism were less pronounced, particularly in the initial period of the experiment. A marked reduction in P-32, Ca-45, and 2C-14-glycine uptake was found in various fractions of the calcified tissues on the 100th day of experimental hypodynamia.

Immunological characterization of the gluten fractions and their hydrolysates from wheat, rye and barley.

PubMed

Rallabhandi, Prasad; Sharma, Girdhari M; Pereira, Marion; Williams, Kristina M

2015-02-18

Gluten proteins in wheat, rye and barley cause celiac disease, an autoimmune disorder of the small intestine, which affects approximately 1% of the world population. Gluten is comprised of prolamin and glutelin. Since avoidance of dietary gluten is the only option for celiac patients, a sensitive gluten detection and quantitation method is warranted. Most regulatory agencies have set a threshold of 20 ppm gluten in foods labeled gluten-free, based on the currently available ELISA methods. However, these methods may exhibit differences in gluten quantitation from different gluten-containing grains. In this study, prolamin and glutelin fractions were isolated from wheat, rye, barley, oats and corn. Intact and pepsin-trypsin (PT)-digested prolamin and glutelin fractions were used to assess their immunoreactivity and gluten recovery by three sandwich and two competitive ELISA kits. The Western blots revealed varied affinity of ELISA antibodies to gluten-containing grain proteins and no reactivity to oat and corn proteins. ELISA results showed considerable variation in gluten recoveries from both intact and PT-digested gluten fractions among different kits. Prolamin fractions showed higher gluten recovery compared to their respective glutelin fractions. Among prolamins, barley exhibited higher recovery compared to wheat and rye with most of the ELISA kits used. Hydrolysis resulted in reduced gluten recovery of most gluten fractions. These results suggest that the suitability of ELISA for accurate gluten quantitation is dependent upon various factors, such as grain source, antibody specificity, gluten proteins and the level of their hydrolysis in foods.

Basic Mathematics Machine Calculator Course.

ERIC Educational Resources Information Center

Windsor Public Schools, CT.

This series of four text-workbooks was designed for tenth grade mathematics students who have exhibited lack of problem-solving skills. Electric desk calculators are to be used with the text. In the first five chapters of the series, students learn how to use the machine while reviewing basic operations with whole numbers, decimals, fractions, and…

Characterization, localization and function of pertussis toxin-sensitive G proteins in the nervous systems of Aplysia and Loligo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogel, S.S.

1989-01-01

The author has characterized pertussis toxin-sensitive G proteins in the nervous systems of the gastropod mollusc Aplysia and the cephalopod Loligo using ({sup 32}P)ADP-ribosylation and immunoblotting with G protein specific antisera. As in vertebrates, this class of G protein is associated with membranes and enriched in nervous tissue in Aplysia. Analysis of dissected Aplysia ganglia reveal that it is enriched in neuropil, a region containing most of the central nervous system synapses. Because both Aplysia and Loligo synaptosomes are enriched in pertussis toxin-sensitive G proteins, it is likely that they are found in synaptic terminals. Fractionation of Aplysia synaptosomes intomore » membrane and vesicle fractions reveals that, although the majority of G protein is recovered in the plasma membrane fraction, a small proportion is recovered in the vesicle fraction. He shows that G proteins are on intracellular membranes by ADP-ribosylating extruded axoplasm with pertussis toxin. A plausible explanation for vesicular localization of G protein in axoplasm is that G proteins are transported to terminals on vesicles. He has shown, using ligature experiments with Aplysia connectives and temperature block experiments in the giant axon of Loligo, that G proteins move by anterograde fast axonal transport. Injection of pertussis toxin into the identified Aplysia neuron L10 blocks histamine-induced presynaptic inhibition of transmitter release. This suggests that pertussis toxin sensitive G proteins play a role in modulating transmitter release at synaptic terminals. In the giant synapse of Loligo, he presents preliminary data that demonstrates that the activation of G proteins in the presynaptic terminal results in decreased transmitter release.« less

Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey.

PubMed

Mohammed, Seif Eldin A; Kabashi, Ahmed S; Koko, Waleed S; Azim, M Kamran

2015-01-01

Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC50 values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents.

Assessment of indigenous Nepalese soybean as a potential germplasm resource for improvement of protein in North American cultivars.

PubMed

Krishnan, Hari B; Natarajan, Savithiry S; Mahmoud, Ahmed A; Bennett, John O; Krishnan, Ammulu Hari; Prasad, Braj Nandan

2006-07-26

Soybeans contain approximately 40% protein and 20% oil and represents an important source of protein in animal rations and human diets. Attempts are being made to increase further the overall protein content of soybeans by utilization of exotic germplasms. In this study, soybean cultivars from Nepal have been characterized and their potential as a germplasm resource for improvement of the protein content and quality of North American cultivars assessed. Soybean cultivars 'Sathia', 'Seti', 'Kavre', and 'Soida Chiny', indigenous to various regions of Nepal, contained 42-45% protein, which is significantly higher in comparison to that of the North American cultivar 'Williams 82' (39%). Fractionation of seed protein by high-resolution two-dimensional gel electrophoresis revealed differences in the protein profiles of these cultivars. Various isoelectric forms of glycinin and beta-conglycinin were identified by comparing the matrix-assisted laser desorption ionization time-of-flight mass fingerprinting data against the National Center for Biotechnology Information nonredundant database. Nepalese cultivar Sathia was distinct, lacking some isoelectric forms of acidic and basic glycinin subunits while expressing other unique forms. The contribution of these unique protein spots present in either Sathia or Williams 82 to the total protein content was quantified using scanning laser densitometry. Distinct restriction fragment length polymorphisms (RFLP) for group 1 glycinin genes were observed among the tested Nepalese genotypes, indicating sequence variation among the cultivars. Conversely, evaluation of RFLP for the genes encoding group 2 glycinins, beta-conglycinin, and Bowman-Birk proteinase inhibitors indicated a high degree of conservation in these genes. Determination of amino acid composition, a reflection of protein quality, indicated that the arginine content of the Nepalese soybeans ranged from 7.7 to 8.1%, which was 5-10% higher than the 7.4% expressed in Williams 82. Additionally, Karve and Seti contained significantly more cysteine than Williams 82. Nepalese high-protein soybeans having a desirable amino acid composition hold potential to increase the protein quality and diversity of North American cultivars.

White clover fractions as protein source for monogastrics: dry matter digestibility and protein digestibility-corrected amino acid scores.

PubMed

Stødkilde, Lene; Damborg, Vinni K; Jørgensen, Henry; Laerke, Helle N; Jensen, Søren K

2018-05-01

The present study aimed to evaluate the use of white clover as an alternative protein source for monogastrics. White clover plant and leaves were processed using a screw-press resulting in a solid pulp and a juice from which protein was acid-precipitated. The chemical composition of all fractions was determined and digestibility of dry matter (DM) and protein was assessed in an experiment with growing rats. Protein concentrates were produced with crude protein (CP) contents of 451 g kg -1 and 530 g kg -1 DM for white clover plant and leaves, respectively, and a pulp with CP contents of 313 and 374 g kg -1 DM from plant and leaves, respectively. The amino acid composition ranged from 4.72 to 6.49 g per 16 g of nitrogen (N) for lysine, 1.82-2.6 g per 16 g N for methionine and cysteine, and 3.66-5.24 g per 16 g N for threonine. True faecal digestibility of protein varied from 0.81 to 0.88, whereas DM digestibility was in the range 0.72-0.80. Methionine and cysteine were found to be limiting in all fractions, regardless of the reference group used. A high digestibility of white clover protein was found irrespective of the physical fractionation. Together with a well-balanced amino acid composition, this makes white clover a promising protein source for monogastrics. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Proteomic analysis of human aqueous humor using multidimensional protein identification technology

PubMed Central

Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

2009-01-01

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

Profiling the Aspergillus fumigatus Proteome in Response to Caspofungin ▿ †

PubMed Central

Cagas, Steven E.; Jain, Mohit Raja; Li, Hong; Perlin, David S.

2011-01-01

The proteomic response of Aspergillus fumigatus to caspofungin was evaluated by gel-free isobaric tagging for relative and absolute quantitation (iTRAQ) as a means to determine potential biomarkers of drug action. A cell fractionation approach yielding 4 subcellular compartment fractions was used to enhance the resolution of proteins for proteomic analysis. Using iTRAQ, a total of 471 unique proteins were identified in soluble and cell wall/plasma membrane fractions at 24 and 48 h of growth in rich media in a wild-type drug-susceptible strain. A total of 122 proteins showed at least a 2-fold change in relative abundance following exposure to caspofungin (CSF) at just below the minimum effective concentration (0.12 μg/ml). The largest changes were seen in the mitochondrial hypoxia response domain protein (AFUA_1G12250), the level of which decreased >16-fold in the secreted fraction, and ChiA1, the level of which decreased 12.1-fold in the cell wall/plasma membrane fraction. The level of the major allergen and cytotoxin AspF1 was also shown to decrease by 12.1-fold upon the addition of drug. A subsequent iTRAQ analysis of an echinocandin-resistant strain (fks1-S678P) was used to validate proteins specific to drug action. A total of 103 proteins in the 2 fractions tested by iTRAQ were differentially expressed in the wild-type susceptible strain but not significantly changed in the resistant strain. Of these potential biomarkers, 11 had levels that changed at least 12-fold. Microarray analysis of the susceptible strain was performed to evaluate the correlation between proteomics and genomics, with a total of 117 genes found to be changing at least 2-fold. Of these, a total of 22 proteins with significant changes identified by iTRAQ also showed significant gene expression level changes by microarray. Overall, these data have the potential to identify biomarkers that assess the relative efficacy of echinocandin drug therapy. PMID:20974863

Characterization and production of multifunctional cationic peptides derived from rice proteins.

PubMed

Taniguchi, Masayuki; Ochiai, Akihito

2017-04-01

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.

DNA and proteins of the nuclear matrix are the main targets of benzo[a]pyrene's action in rat hepatocytes.

PubMed

Widłak, P; Rzeszowska-Wolny, J

1993-01-01

The binding of [14C]benzo[a]pyrene (B[a]P) to DNA and proteins in total nuclei and subnuclear fractions of cultured rat hepatocytes was compared. The main targets of B[a]P were non-histone high molecular weight proteins of the nuclear matrix and DNA sequences attached to this structure. Following 24 h exposure to B[a]P the amounts of adducts in the nuclear matrix DNA and proteins were twice as high as in total nuclei. After withdrawal of the carcinogen containing medium the level of B[a]P-induced adducts gradually decreased but always remained the highest in the nuclear matrix proteins. Removal of adducts from the nuclear matrix DNA was more efficient than from the other DNA fractions, and 72 h after exposure to the carcinogen the level of DNA adducts in this fraction was similar to that in total nuclei.

Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

PubMed Central

Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

2015-01-01

The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

Nuclear matrix and hnRNP share a common structural constituent associated with premessenger RNA.

PubMed Central

Gallinaro, H; Puvion, E; Kister, L; Jacob, M

1983-01-01

Nuclear matrix and heterogeneous nuclear ribonucleoprotein (hnRNP) were compared to establish whether premessenger RNA (premRNA) was associated with a same constituent in both structures. The isolation of nuclear matrix included the removal of chromatin and of 0.4 M KCl-soluble material. HnRNP, isolated by a standard method was also treated by 0.4 M KCl. Both isolation procedures caused the removal of DNA, histones, a fraction of small nuclear RNA and of nonhistone proteins including the hnRNP proteins in the 30 000-40 000 mol. wt. range. High resolution autoradiography showed that hnRNA remained associated with the residual fibrils in both structures. They both contained the same premRNA and maturation products as shown by the analysis of the transcripts of the early region 3 of adenovirus 2. In addition, the small nuclear RNA and protein of the salt-resistant complexes were also present in the matrix. The results are compatible with the idea that the salt-resistant complexes from hnRNP constitute the fibrils associated with premRNA in the nucleoplasmic matrix. The fibrils may be the basic unit of splicing and their organization in matrix might provide the spatial configuration necessary for regulation. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. PMID:6557026

Electricity generation from food wastes and characteristics of organic matters in microbial fuel cell.

PubMed

Li, Hui; Tian, Yu; Zuo, Wei; Zhang, Jun; Pan, Xiaoyue; Li, Lipin; Su, Xinying

2016-04-01

The microbial fuel cell (MFC) was evaluated as an alternative way to recover electricity from canteen based food waste. Characteristics of the organics in food waste before and after the MFC treatment were analyzed to investigate how the organic matters were biodegraded and transformed during the MFC treatment. A maximum power density of 5.6W/m(3) and an average output voltage of 0.51V were obtained. During the MFC operation, the hydrophilic and acidic fractions were more readily degraded, compared to the neutral fractions. Additionally, aromatic compounds in the hydrophilic fraction were more preferentially removed than non-aromatic compounds. The MFC could easily remove the tryptophan protein-like substances in all fractions and aromatic proteins in hydrophilic and hydrophobic neutral fractions. Additionally, the hydrophobic amide-1 proteins and aliphatic components were readily hydrolyzed and biodegraded in the MFC. These findings may facilitate the pretreatment and posttreatment choices for MFC system fed with food waste. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of non-ideal analyte behavior on the separation of protein aggregates by asymmetric flow field-flow fractionation.

PubMed

Boll, Björn; Josse, Lena; Heubach, Anja; Hochenauer, Sophie; Finkler, Christof; Huwyler, Jörg; Koulov, Atanas V

2018-04-25

Asymmetric flow field-flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field-flow fractionation is non-trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here we report methods to assess the non-ideal effects that influence asymmetric flow field-flow fractionation separation and for the first time identify experimentally the main factors that impact it. Furthermore, we propose new approaches to minimize such non-ideal behavior, showing that by adjusting the mobile phase composition (pH and ionic strength) the resolution of asymmetric flow field-flow fractionation separation can be drastically improved. Additionally, we propose a best practice method for new proteins. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture

PubMed Central

Jaquinod, Michel; Villiers, Florent; Kieffer-Jaquinod, Sylvie; Hugouvieux, Véronique; Bruley, Christophe; Garin, Jérôme; Bourguignon, Jacques

2007-01-01

To better understand the mechanisms governing cellular traffic, storage of various metabolites and their ultimate degradation, Arabidopsis thaliana vacuoles proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker α-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42 fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomic study. Therefore, a proteomic approach was developed in order to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, (iii) a pre-fractionation of proteins by short migration on SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, 2/3 of which copurify with the membrane hydrophobic fraction and 1/3 with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were previously known to be associated with vacuolar activities. The proteins identified are involved in: ion and metabolite transport (26%), stress response (9%), signal transduction (7%), metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein- and sugar-hydrolysis. The sub-cellular localization of several putative vacuolar proteins was confirmed by transient expression of GFP-fusion constructs. PMID:17151019

Requirement of the basic region of N-WASP/WAVE2 for actin-based motility.

PubMed

Suetsugu, S; Miki, H; Yamaguchi, H; Takenawa, T

2001-04-06

WASP family proteins activate nucleation by the Arp2/3 complex, inducing rapid actin polymerization in vitro. Although the C-terminal portion of WASP family proteins (VCA) activates nucleation by the Arp2/3 complex in pure systems, we find that this fragment lacks activity in cell extracts. Thus, polystyrene beads coated with VCA did not move in brain cytosol, while beads coated with N-WASP or WAVE2 did move. The basic clusters between the WH1 domain and the CRIB domain of N-WASP were critical for movement since beads coated with N-WASP or WAVE2 constructs missing the basic clusters (Delta basic) also did not move. Furthermore, VCA and N-WASP/WAVE2 Delta basic constructs were much less able than wild-type N-WASP and WAVE2 to induce actin polymerization in cytosol. All of the proteins, with or without the basic domain, were potent activators of nucleation by purified Arp2/3 complex. Copyright 2001 Academic Press.

Impact of ultrafiltration and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties.

PubMed

Picot, Laurent; Ravallec, Rozenn; Fouchereau-Péron, Martine; Vandanjon, Laurent; Jaouen, Pascal; Chaplain-Derouiniot, Maryse; Guérard, Fabienne; Chabeaud, Aurélie; Legal, Yves; Alvarez, Oscar Martinez; Bergé, Jean-Pascal; Piot, Jean-Marie; Batista, Irineu; Pires, Carla; Thorkelsson, Gudjon; Delannoy, Charles; Jakobsen, Greta; Johansson, Inez; Bourseau, Patrick

2010-08-30

Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation. Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature. Copyright (c) 2010 Society of Chemical Industry.

Comparison of Gluten Extraction Protocols Assessed by LC-MS/MS Analysis.

PubMed

Fallahbaghery, Azadeh; Zou, Wei; Byrne, Keren; Howitt, Crispin A; Colgrave, Michelle L

2017-04-05

The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology (antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple "IPA/DTT" protocol and related "two-step" protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.

Albizia lebbeck Seed Coat Proteins Bind to Chitin and Act as a Defense against Cowpea Weevil Callosobruchus maculatus.

PubMed

Silva, Nadia C M; De Sá, Leonardo F R; Oliveira, Eduardo A G; Costa, Monique N; Ferreira, Andre T S; Perales, Jonas; Fernandes, Kátia V S; Xavier-Filho, Jose; Oliveira, Antonia E A

2016-05-11

The seed coat is an external tissue that participates in defense against insects. In some nonhost seeds, including Albizia lebbeck, the insect Callosobruchus maculatus dies during seed coat penetration. We investigated the toxicity of A. lebbeck seed coat proteins to C. maculatus. A chitin-binding protein fraction was isolated from seed coat, and mass spectrometry showed similarity to a C1 cysteine protease. By ELM program an N-glycosylation interaction motif was identified in this protein, and by molecular docking the potential to interact with N-acetylglucosamine (NAG) was shown. The chitin-binding protein fraction was toxic to C. maculatus and was present in larval midgut and feces but not able to hydrolyze larval gut proteins. It did not interfere, though, with the intestinal cell permeability. These results indicate that the toxicity mechanism of this seed coat fraction may be related to its binding to chitin, present in the larvae gut, disturbing nutrient absorption.

Influence of the yeast strain on the changes of the amino acids, peptides and proteins during sparkling wine production by the traditional method.

PubMed

Martínez-Rodríguez, A J; Carrascosa, A V; Martín-Alvarez, P J; Moreno-Arribas, V; Polo, M C

2002-12-01

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions.

Bioaccessibility of Ca, Cu, Fe, Mg, Zn, and crude protein in beef, pork and chicken after thermal processing.

PubMed

Menezes, Eveline A; Oliveira, Aline F; França, Celia J; Souza, Gilberto B; Nogueira, Ana Rita A

2018-02-01

The bioaccessibility of Ca, Cu, Fe, Mg, Zn, and crude protein was evaluated after submitting beef, pork, and chicken to five different thermal treatments. The bioaccessibility of crude protein and metals were simulated by using in vitro enzymatic digestion with a gastric fluid solution and dialysability approach. Inductively coupled plasma optical spectrometry was used to quantify the dialyzable fraction and the total mineral content after microwave-assisted digestion. Graphite furnace atomic absorption spectrometry quantified Cu in chicken dialyzable fraction. The increase of temperature and heat exposure period decreased the protein bioaccessibility. Considering the total and dialyzable fraction, beef is an important source of Cu, Fe, Mg, and Zn to the human diet. The results of Fourier-transform infrared spectroscopy indicated physical changes in the treated samples related to protein denaturation, which was probably responsible for the decreased bioaccessibility of minerals and protein, mainly at higher temperatures. Copyright © 2017 Elsevier Ltd. All rights reserved.

Off-Line Multidimensional Liquid Chromatography and Auto Sampling Result in Sample Loss in LC/LC–MS/MS

PubMed Central

2015-01-01

Large-scale proteomics often employs two orthogonal separation methods to fractionate complex peptide mixtures. Fractionation can involve ion exchange separation coupled to reversed-phase separation or, more recently, two reversed-phase separations performed at different pH values. When multidimensional separations are combined with tandem mass spectrometry for protein identification, the strategy is often referred to as multidimensional protein identification technology (MudPIT). MudPIT has been used in either an automated (online) or manual (offline) format. In this study, we evaluated the performance of different MudPIT strategies by both label-free and tandem mass tag (TMT) isobaric tagging. Our findings revealed that online MudPIT provided more peptide/protein identifications and higher sequence coverage than offline platforms. When employing an off-line fractionation method with direct loading of samples onto the column from an eppendorf tube via a high-pressure device, a 5.3% loss in protein identifications is observed. When off-line fractionated samples are loaded via an autosampler, a 44.5% loss in protein identifications is observed compared with direct loading of samples onto a triphasic capillary column. Moreover, peptide recovery was significantly lower after offline fractionation than in online fractionation. Signal-to-noise (S/N) ratio, however, was not significantly altered between experimental groups. It is likely that offline sample collection results in stochastic peptide loss due to noncovalent adsorption to solid surfaces. Therefore, the use of the offline approaches should be considered carefully when processing minute quantities of valuable samples. PMID:25040086

Protein disorder is positively correlated with gene expression in E. coli

PubMed Central

Paliy, Oleg; Gargac, Shawn M.; Cheng, Yugong; Uversky, Vladimir N.; Dunker, A. Keith

2009-01-01

We considered on a global scale the relationship between the predicted fraction of protein disorder and RNA and protein expression in E. coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins. PMID:18465893

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samarel, A.M.; Parmacek, M.S.; Magid, N.M.

To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of (/sup 3/H) leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesismore » (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting.« less

Teaching Stable Two-Mirror Resonators through the Fractional Fourier Transform

ERIC Educational Resources Information Center

Moreno, Ignacio; Garcia-Martinez, Pascuala; Ferreira, Carlos

2010-01-01

We analyse two-mirror resonators in terms of their fractional Fourier transform (FRFT) properties. We use the basic ABCD ray transfer matrix method to show how the resonator can be regarded as the cascade of two propagation-lens-propagation FRFT systems. Then, we present a connection between the geometric properties of the resonator (the g…

Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster.

PubMed

Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Walter, Frederik; Wolf, Timo; Zemke, Till; Wehmeier, Udo F; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

2015-07-01

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane. Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species. Copyright © 2015 Elsevier B.V. All rights reserved.

Estimating conformation content of a protein using citrate-stabilized Au nanoparticles

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Paul, Anumita; Chattopadhyay, Arun

2010-08-01

Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration. Electronic supplementary information (ESI) available: Additional UV-vis and fluorescence spectra and graphs based on UV-vis studies. See DOI: 10.1039/c0nr00154f

Inhibitory effects of ethyl acetate extract of Teucrium polium on in vitro protein glycoxidation.

PubMed

Ardestani, Amin; Yazdanparast, Razieh

2007-12-01

Regarding the involvement of free radicals and oxidative reactions in protein glycoxidation processes, compounds with antioxidant activities have been tested in order to reduce or to stop glycoxidation. In this study, we evaluated the antioxidant potential of several organic fractions of Teucrium polium extract using different model systems including total antioxidant capacity by the phosphomolybdenum method, ferric reducing antioxidant power and Trolox equivalent antioxidant capacity assays, antioxidant activity in linoleic acid emulsion system and scavenging of 1,1-diphenyl-2-picrylhydrazyl radical. The results indicated that the ethyl acetate (EtOAc) fraction of T. polium possesses the highest antioxidant activity and total phenolic and flavonoid contents. Given the link between glycation and oxidation, we proposed that the EtOAc fraction might possess significant in vitro antiglycation activities as well. Our data confirmed the inhibitory effect of EtOAc fraction on bovine serum albumin (BSA) glycoxidation measured in terms of advanced glycation end products (AGEs) and pentosidine formation as well as protein oxidation markers including protein carbonyl formation (PCO) and loss of protein thiols. Reducing sugars such as ribose and glucose increase fluorescence intensity of glycated BSA in terms of total AGEs and pentosidine during 21 day of exposure. Moreover, sugars cause more PCO formation and also oxidize thiol groups more in glycated than in native BSA. EtOAc extract at different concentrations (10-100 microg/ml) has significantly quenched the fluorescence intensity of glycated BSA. Furthermore, we demonstrated that the inhibitory effect of EtOAc extract in preventing oxidative protein damages including effect on PCO formation and thiol oxidation which are believe to form under the glycoxidation process. These results clearly demonstrate that, the EtOAc fraction, owning to its antioxidant content, is capable of suppressing the formation of AGEs and protein oxidation in vitro.

Exact soliton of (2 + 1)-dimensional fractional Schrödinger equation

NASA Astrophysics Data System (ADS)

Rizvi, S. T. R.; Ali, K.; Bashir, S.; Younis, M.; Ashraf, R.; Ahmad, M. O.

2017-07-01

The nonlinear fractional Schrödinger equation is the basic equation of fractional quantum mechanics introduced by Nick Laskin in 2002. We apply three tools to solve this mathematical-physical model. First, we find the solitary wave solutions including the trigonometric traveling wave solutions, bell and kink shape solitons using the F-expansion and Improve F-expansion method. We also obtain the soliton solution, singular soliton solutions, rational function solution and elliptic integral function solutions, with the help of the extended trial equation method.

Fractional Talbot field and of finite gratings: compact analytical formulation.

PubMed

Arrizón, V; Rojo-Velázquez, G

2001-06-01

We present a compact analytical formulation for the fractional Talbot effect at the paraxial domain of a finite grating. Our results show that laterally shifted distorted images of the grating basic cell form the Fresnel field at a fractional Talbot plane of the grating. Our formulas give the positions of those images and show that they are given by the convolution of the nondistorted cells (modulated by a quadratic phase factor) with the Fourier transform of the finite-grating pupil.

[Properties and localization of Mg- and Ca-ATpase activities in wheat embryo cell nuclei].

PubMed

Vasil'eva, N A; Belkina, G G; Stepanenko, S Y; Atalykova, F I; Oparin, A I

1978-05-01

The isolated nuclei of wheat embryo possess the ATPase activity. The addition of Mg2+ and Ca2+ significantly increases the activities of nuclear ATPases, whereas Hg2+, Cu2+ and Mn2+ inhibit the activity. The activating effect of Mg2+ is enhanced by an addition of Na and K ions. The activity of wheat embryo nuclear Mg-ATPase is higher than its Ca-ATPase activity; both ATPases also differ in their pH optima. Separation of total nuclear protein according to the solubility of its individual protein components in wheat and strong salt solutions, using the detergents, as well as ammonium sulfate precipitation and dialysis do not result in separation of Mg-activated and Ca-activated ATPases, although their levels of activities and ratios change in the course of fractionation. The Mg- and Ca-ATPase activities of the wheat embryo nuclei were found in the nuclear fraction of albumin, in nonhistone proteins and nuclear membranes. In the albumin nuclear fraction and subfractions of non-histone proteins the higher level of activity is observed in Ca-ATPase, whereas in the nuclei and soluble fractions of residual proteins in Mg-ATPase.

Enrichment and Purification of Casein Glycomacropeptide from Whey Protein Isolate Using Supercritical Carbon Dioxide Processing and Membrane Ultrafiltration

PubMed Central

Bonnaillie, Laetitia M.; Qi, Phoebe; Wickham, Edward; Tomasula, Peggy M.

2014-01-01

Whey protein concentrates (WPC) and isolates (WPI), comprised mainly of β-lactoglobulin (β-LG), α-lactalbumin (α-LA) and casein glycomacropeptide (GMP), are added to foods to boost nutritional and functional properties. Supercritical carbon dioxide (SCO2) has been shown to effectively fractionate WPC and WPI to obtain enriched fractions of α-LA and β-LG, thus creating new whey ingredients that exploit the properties of the individual component proteins. In this study, we used SCO2 to further fractionate WPI via acid precipitation of α-LA, β-LG and the minor whey proteins to obtain GMP-enriched solutions. The process was optimized and α-LA precipitation maximized at low pH and a temperature (T) ≥65 °C, where β-LG with 84% purity and GMP with 58% purity were obtained, after ultrafiltration and diafiltration to separate β-LG from the GMP solution. At 70 °C, β-LG also precipitated with α-LA, leaving a GMP-rich solution with up to 94% purity after ultrafiltration. The different protein fractions produced with the SCO2 process will permit the design of new foods and beverages to target specific nutritional needs. PMID:28234306

Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

PubMed

Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

1990-03-01

We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.

Antioxidant activities of bambara groundnut (Vigna subterranea) protein hydrolysates and their membrane ultrafiltration fractions.

PubMed

Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Ijabadeniyi, Oluwatosin A; Aluko, Rotimi E; Amonsou, Eric O

2016-05-18

In this study, the bambara protein isolate (BPI) was digested with three proteases (alcalase, trypsin and pepsin), to produce bambara protein hydrolysates (BPHs). These hydrolysates were passed through ultrafiltration membranes to obtain peptide fractions of different sizes (<1, 1-3, 3-5 and 5-10 kDa). The hydrolysates and their peptide fractions were investigated for antioxidant activities. The membrane fractions showed that peptides with sizes <3 kDa had significantly (p < 0.05) reduced surface hydrophobicity when compared with peptides >3 kDa. This is in agreement with the result obtained for the ferric reducing power, metal chelating and hydroxyl radical scavenging activities where higher molecular weight peptides exhibited better activity (p < 0.05) when compared to low molecular weight peptide fractions. However, for all the hydrolysates, the low molecular weight peptides were more effective diphenyl-1-picrylhydrazyl (DPPH) radical scavengers but not superoxide radicals when compared to the bigger peptides. In comparison with glutathione (GSH), BPHs and their membrane fractions had better (p < 0.05) reducing power and ability to chelate metal ions except for the pepsin hydrolysate and its membrane fractions that did not show any metal chelating activity. However, the 5-10 kDa pepsin hydrolysate peptide fractions had greater (88%) hydroxyl scavenging activity than GSH, alcalase and trypsin hydrolysates (82%). These findings show the potential use of BPHs and their peptide fraction as antioxidants in reducing food spoilage or management of oxidative stress-related metabolic disorders.

Toward understanding the role of individual fluorescent components in DOM-metal binding.

PubMed

Wu, Jun; Zhang, Hua; Yao, Qi-Sheng; Shao, Li-Ming; He, Pin-Jing

2012-05-15

Knowledge on the function of individual fractions in dissolved organic matter (DOM) is essential for understanding the impact of DOM on metal speciation and migration. Herein, fluorescence excitation-emission matrix quenching and parallel factor (PARAFAC) analysis were adopted for bulk DOM and chemically isolated fractions from landfill leachate, i.e., humic acids (HA), fulvic acids and hydrophilic (HyI) fraction, to elucidate the role of individual fluorescent components in metal binding (Cu(II) and Cd(II)). Three components were identified by PARAFAC model, including one humic substance (HS)-like, one protein-like and one component highly correlated with the HyI fraction. Among them, the HS-like and protein-like components were responsible for Cu(II) binding, while the protein-like component was the only fraction involved in Cd(II) complexation. It was further identified that the slight quenching effect of HA fraction by Cd(II) was induced by the presence of proteinaceous materials in HA. Fluorescent substances in the HyI fraction of landfill leachate did not play as important a role as HS did. Therefore, it was suggested that the potential risk of aged leachate (more humified) as a carrier of heavy metal should not be overlooked. Copyright © 2012 Elsevier B.V. All rights reserved.

Wet-milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine.

PubMed

Moeller, Lorena; Taylor-Vokes, Raye; Fox, Steve; Gan, Qinglei; Johnson, Lawrence; Wang, Kan

2010-01-01

The production of recombinant proteins in plants continues to be of great interest for prospective large-scale manufacturing of industrial enzymes, nutrition products, and vaccines. This work describes fractionation by wet-milling of transgenic maize expressing the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B), a potent immunogen and candidate for oral vaccine and vaccine components. The LT-B gene was directed to express in seed by an endosperm specific promoter. Two steeping treatments, traditional steeping (TS, 0.2% SO(2) + 0.5% lactic acid) and water steeping (WS, water only), were evaluated to determine effects on recovery of functional LT-B in wet-milled fractions. The overall recovery of the LT-B protein from WS treatment was 1.5-fold greater than that from TS treatment. In both steeping types, LT-B was distributed similarly among the fractions, resulting in enrichment of functional LT-B in fine fiber, coarse fiber and pericarp fractions by concentration factors of 1.5 to 8 relative to the whole kernels on a per-mass basis. Combined with endosperm-specific expression and secretory pathway targeting, wet-milling enables enrichment of high-value recombinant proteins in low-value fractions, such as the fine fiber, and co-utilization of remaining fractions in alternative industrial applications.

Comparison of biochemical properties of liver arginase from streptozocin-induced diabetic and control mice.

PubMed

Spolarics, Z; Bond, J S

1989-11-01

Arginase activity is elevated in livers of diabetic animals compared to controls and there is evidence that this is due in part to increased specific activity (activity/mg arginase protein). To investigate the molecular basis of this increased activity, the physicochemical and kinetic properties of hepatic arginase from diabetic and control mice were compared. Two types of arginase subunits with molecular weights of 35,000 and 38,000 were found in both the diabetic and control animals and the subunits in these animals had similar, multiple ionic forms. Kinetic parameters of purified preparations of arginase for arginine (apparent Km and Vmax values) and the thermal stability of these preparations from diabetics and controls were also similar. Furthermore, no difference was found in the distribution of arginase activity among different subcellular liver fractions. Separation of basic and acidic oligomeric forms of arginase by fast-protein liquid chromatography resulted in a slightly different distribution of activity among the forms in the normal and diabetic group. The apparent Km values for Mn2+ of the basic form of the enzyme were 25 and 33 microM for the enzyme from normal and diabetic animals, respectively; for acidic forms, for which two apparent Km values were measured, the values were 8 and 197 microM for arginase from controls and 35 and 537 microM from diabetics. These results indicate that in diabetes, while no marked changes in the physicochemical characteristics of arginase are obvious, some changes are found in the interaction of arginase with its cofactor Mn.

High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

PubMed

Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

2014-07-01

Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Effect of restricted forage intake on ruminal disappearance of bromegrass hay and a blood meal, feather meal, and fish meal supplement.

PubMed

Scholljegerdes, E J; Ludden, P A; Hess, B W

2005-09-01

Two experiments were conducted to determine in situ disappearance of bromegrass hay and a ruminally undegraded protein (RUP) supplement in beef cattle fed restricted amounts of forage. Six Angus crossbred cattle (BW = 589 +/- 44.4 kg; three steers and three heifers) fitted with ruminal cannulas were fed chopped (2.54 cm) bromegrass hay (8.9% CP) at one of three percentages of maintenance intake (30, 55, or 80%; one steer and one heifer per treatment). In both experiments, the cattle were allowed 7 d for diet adaptation followed by 3 d of sample collection. In Exp 1, in situ bags (50 microm pore size) containing 4.1 g of brome-grass hay (OM basis) were inserted into the rumen and subsequently removed at 3, 6, 9, 12, 15, 18, 24, 36, and 48 h after insertion. Nonlinear regression models were used to determine the rapidly solubilized protein Fraction A, the potentially ruminal degradable protein Fraction B, the ruminally undegraded protein Fraction C, and protein degradation rate. Intake level did not affect (P = 0.15 to 0.95) forage protein remaining after in situ incubation or Fractions A, B, and C; however, effective ruminal degradation of hay protein tended to increase quadratically (P = 0.12) as forage intake increased. In Exp 2, 4.2 g (OM basis) of an RUP supplement (6.8% porcine blood meal, 24.5% hydrolyzed feather meal, and 68.7% menhaden fish meal) formulated to provide equal amounts of metabolizable protein across all levels of hay consumption was evaluated in a similar manner as in Exp 1. The undegraded protein fraction of the supplement did not differ (P = 0.16 to 0.74) across treatments at 3, 6, 9, and 18 h; however, increasing forage intake resulted in a linear increase (P < or = 0.06) in undegraded protein remaining at 12, 15, 24, 36, and 48 h. Dietary treatment had no affect (P = 0.30) on protein Fractions A, B, or C; however, protein degradation rate of the supplement decreased linearly (P = 0.03) as forage intake increased. Therefore, effective ruminal degradation of the supplement decreased linearly (P = 0.01) from 50.8 to 40.9% as forage intake increased from 30 to 80% of maintenance. Corresponding estimates of supplement RUP were 49.2, 56.5, and 59.1% for the 30, 55, and 80% of maintenance intake treatments, respectively. Restricting dietary intake can decrease the quantity of dietary protein that escapes ruminal degradation. Tabular estimates of RUP may not be appropriate for formulating diets to balance metabolizable protein in beef cattle consuming limited quantities of forage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giannone, Richard J.; Wurch, Louie L.; Podar, Mircea

The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, includingmore » membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.« less

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stekhoven, Daniel J.; Omasits, Ulrich; Quebatte, Maxime

2014-03-01

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled usmore » to distinguish cytoplasmic, peripheral innermembrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion.« less

Survival of Escherichia coli after isoelectric solubilization and precipitation of fish protein.

PubMed

Lansdowne, L R; Beamer, S; Jaczynski, J; Matak, K E

2009-07-01

Protein recovery for fish processing by-products utilizes extreme pH shifts for isoelectric solubilization and precipitation. The purpose of this study was to determine if Escherichia coli would survive exposure to the extreme pH shifts during the protein recovery process. Fresh rainbow trout were beheaded, gutted, and minced and then inoculated with approximately 10(9) CFU of E. coli ATCC 25922 per g, homogenized, and brought to the target pH of 2.0, 3.0, 11.5, or 12.5 by the addition of concentrated hydrochloric acid or sodium hydroxide to solubilize muscle proteins. The homogenate was blended and centrifuged to separate the lipid and insoluble components (bones, skin, insoluble protein, etc.) from the protein solution. The protein solution was subjected to a second pH shift (pH 5.5) resulting in protein precipitation that was recovered with centrifugation. Microbial analysis was conducted on each fraction (i.e., lipid, insoluble components, protein, and water) with selective and nonselective media. The sums of the surviving E. coli in these fractions were compared with the initial inoculum. The greatest total microbial reduction occurred when the pH was shifted to 12.5 (P < 0.05), i.e., a 4.4-log reduction of cells on nonselective media and a 6.0-log reduction of cells on selective media. The use of selective and nonselective media showed that there was significant (P < 0.05) injury sustained by cells exposed to alkaline treatment (pH 11.5 and 12.5) in all fractions except the insoluble fraction at pH 11.5. Increasing the exposure time or the pH may result in greater bacterial reductions in the recovered protein.

Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rong, Hui; Li, Yan; Lou, Xiao-hua

2007-02-01

A novel cardiotoxin-like basic protein from Naja naja atra was crystallized and diffraction data were collected to 2.35 Å resolution. A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data setmore » was collected to 2.35 Å resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 43.2, c = 147.9 Å. There are two molecules in the crystallographic asymmetric unit.« less

Thermoase-Derived Flaxseed Protein Hydrolysates and Membrane Ultrafiltration Peptide Fractions Have Systolic Blood Pressure-Lowering Effects in Spontaneously Hypertensive Rats

PubMed Central

Nwachukwu, Ifeanyi D.; Girgih, Abraham T.; Malomo, Sunday A.; Onuh, John O.; Aluko, Rotimi E.

2014-01-01

Thermoase-digested flaxseed protein hydrolysate (FPH) samples and ultrafiltration membrane-separated peptide fractions were initially evaluated for in vitro inhibition of angiotensin I-converting enzyme (ACE) and renin activities. The two most active FPH samples and their corresponding peptide fractions were subsequently tested for in vivo antihypertensive activity in spontaneously hypertensive rats (SHR). The FPH produced with 3% thermoase digestion showed the highest ACE- and renin-inhibitory activities. Whereas membrane ultrafiltration resulted in significant (p < 0.05) increases in ACE inhibition by the <1 and 1–3 kDa peptides, only a marginal improvement in renin-inhibitory activity was observed for virtually all the samples after membrane ultrafiltration. The FPH samples and membrane fractions were also effective in lowering systolic blood pressure (SBP) in SHR with the largest effect occurring after oral administration (200 mg/kg body weight) of the 1–3 kDa peptide fraction of the 2.5% FPH and the 3–5 kDa fraction of the 3% FPH. Such potent SBP-lowering capacity indicates the potential of flaxseed protein-derived bioactive peptides as ingredients for the formulation of antihypertensive functional foods and nutraceuticals. PMID:25302619

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

PubMed

Mangas, I; Vilanova, E; Benabent, M; Estévez, J

2014-02-10

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

[Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

PubMed

Fedina, A B; Gazarian, G G

1976-01-01

Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth.

PubMed

Giannone, Richard J; Wurch, Louie L; Podar, Mircea; Hettich, Robert L

2015-08-04

The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. As this interaction is thought to be membrane-associated, involving a myriad of membrane-anchored proteins, proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitans proteins. Using this method, we show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. These gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.

A Tutorial Review on Fractal Spacetime and Fractional Calculus

NASA Astrophysics Data System (ADS)

He, Ji-Huan

2014-11-01

This tutorial review of fractal-Cantorian spacetime and fractional calculus begins with Leibniz's notation for derivative without limits which can be generalized to discontinuous media like fractal derivative and q-derivative of quantum calculus. Fractal spacetime is used to elucidate some basic properties of fractal which is the foundation of fractional calculus, and El Naschie's mass-energy equation for the dark energy. The variational iteration method is used to introduce the definition of fractional derivatives. Fractal derivative is explained geometrically and q-derivative is motivated by quantum mechanics. Some effective analytical approaches to fractional differential equations, e.g., the variational iteration method, the homotopy perturbation method, the exp-function method, the fractional complex transform, and Yang-Laplace transform, are outlined and the main solution processes are given.

Fractional Klein-Gordon equation composed of Jumarie fractional derivative and its interpretation by a smoothness parameter

NASA Astrophysics Data System (ADS)

Ghosh, Uttam; Banerjee, Joydip; Sarkar, Susmita; Das, Shantanu

2018-06-01

Klein-Gordon equation is one of the basic steps towards relativistic quantum mechanics. In this paper, we have formulated fractional Klein-Gordon equation via Jumarie fractional derivative and found two types of solutions. Zero-mass solution satisfies photon criteria and non-zero mass satisfies general theory of relativity. Further, we have developed rest mass condition which leads us to the concept of hidden wave. Classical Klein-Gordon equation fails to explain a chargeless system as well as a single-particle system. Using the fractional Klein-Gordon equation, we can overcome the problem. The fractional Klein-Gordon equation also leads to the smoothness parameter which is the measurement of the bumpiness of space. Here, by using this smoothness parameter, we have defined and interpreted the various cases.

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

PubMed Central

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-01-01

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

PubMed

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-05-05

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

PubMed Central

Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra

2014-01-01

ABSTRACT Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins. PMID:25078698

Amino acid availability regulates the effect of hyperinsulinemia on skin protein metabolism in pigs

USDA-ARS?s Scientific Manuscript database

The effects of amino acid supply and insulin infusion on skin protein kinetics (fractional synthesis rate (FSR), fractional breakdown rate (FBR), and net balance (NB)) in pigs were investigated. Four-month-old pigs were divided into four groups as follows: control, insulin (INS), amino acid (AA), an...

Distribution of Animal Drugs among Curd, Whey, and Milk Protein Fractions in Spiked Skim Milk and Whey.

PubMed

Shappell, Nancy W; Shelver, Weilin L; Lupton, Sara J; Fanaselle, Wendy; Van Doren, Jane M; Hakk, Heldur

2017-02-01

It is important to understand the partitioning of drugs in processed milk and milk products, when drugs are present in raw milk, in order to estimate the potential consumer exposure. Radioisotopically labeled erythromycin, ivermectin, ketoprofen, oxytetracycline, penicillin G, sulfadimethoxine, and thiabendazole were used to evaluate the distribution of animal drugs among rennet curd, whey, and protein fractions from skim cow milk. Our previous work reported the distribution of these same drugs between skim and fat fractions of milk. Drug distribution between curd and whey was significantly correlated (R 2 = 0.70) to the drug's lipophilicity (log P), with improved correlation using log D (R 2 = 0.95). Distribution of drugs was concentration independent over the range tested (20-2000 nM). With the exception of thiabendazole and ivermectin, more drug was associated with whey protein than casein on a nmol/g protein basis (oxytetracycline experiment not performed). These results provide insights into the distribution of animal drug residues, if present in cow milk, among milk fractions, with possible extrapolation to milk products.

Fractionation and identification of the allergic proteins in Aspergillus species.

PubMed

Falahati, M; Ghanbari, S; Ebrahimi, M; Ghazanfari, M; Bazrafshan, F; Farahyar, S; Falak, R

2016-12-01

Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus , A. flavus , and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity. Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods. Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase. Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.

Effect of mechanical denaturation on surface free energy of protein powders.

PubMed

Mohammad, Mohammad Amin; Grimsey, Ian M; Forbes, Robert T; Blagbrough, Ian S; Conway, Barbara R

2016-10-01

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and β-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of β-galactosidase was higher than that of lysozyme. Also, the surface of β-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (β-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms. Copyright © 2016 Elsevier B.V. All rights reserved.

What Works Clearinghouse Quick Review: "Academic Music: Music Instruction to Engage Third-Grade Students in Learning Basic Fraction Concepts"

ERIC Educational Resources Information Center

What Works Clearinghouse, 2012

2012-01-01

The study examined the effectiveness of an intervention designed to teach mathematical concepts through music. Specifically, it investigated the effect of the intervention on third-grade students' understanding of fractions. Sixty-seven students from one northern California elementary school participated in the study over a period of six weeks; of…

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

PubMed Central

Rao, Marie Luise; Rao, Govind S.

1982-01-01

1. Binding of l-tri-[125I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25°C. 2. The l-tri-[125I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100°C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at −38°C for several months. 3. It displayed saturability, high affinity (apparent Kd 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3′-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[125I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304±0.11μg/mg of cytosol protein (mean±s.d., n=4) was obtained; the mean concentration in plasma was 0.309±0.07μg/mg of plasma protein (mean±s.d., n=3). 7. The Ka value of 6.3×108m−1 of l-tri-[125I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[125I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin. PMID:6289813

[Gluten--mechanisms of intolerance, symptoms and treatment possibilities of IgE-related allergy for gluten in the light of actual clinical and immunological studies].

PubMed

Obtułowicz, Krystyna; Waga, Jacek; Dyga, Wojciech

2015-01-01

Gluten is the product of a chemical bond of wheat prolamin proteins (glia- dins and glutenins) in an aqueous me- dium. IgE mediated gluten allergy can be induced either by gluten as an in- gredient in foods or wheat prolamines present in the air. The aim of the study was clinical analysis of 13 patients, who demonstrated elevated levels of gluten specific IgE and identification of the most allergenic protein fractions from several samples of wheat using serum of examined subjects. Clinical analysis showed the occupational allergy to gluten in the form of rhinitis, asthma and airborne dermatistis in 9 subjects, whose symptoms disappeared during isolation from occupational exposure despite the use of a normal diet. In case of 4 patients with severe forms of chronic urticaria and atopic dermatitis, who are also allergic to grass pollen at the same time, the introduction of a gluten-free diet resulted in improvement of health conditions. The study of wheat protein fractions revealed a significant polymorphism dependent on the wheat sample. In the protein fractions, low and high molecular glutenin fractions, and alpha, beta, gamma, and omega-gliadins were separated. It has been shown that the strongest immunogenic effect causes omega-5 gliadin fraction. The removal of this fraction resulted in reduction of skin reactivity evaluated by skin prick test in the studied patients.

Identification of Potent ACE Inhibitory Peptides from Wild Almond Proteins.

PubMed

Mirzapour, Mozhgan; Rezaei, Karamatollah; Sentandreu, Miguel Angel

2017-10-01

In this study, the production, fractionation, purification and identification of ACE (angiotensin-I-converting enzyme) inhibitory peptides from wild almond (Amygdalus scoparia) proteins were investigated. Wild almond proteins were hydrolyzed using 5 different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme) and assayed for their ACE inhibitory activities. The degree of ACE inhibiting activity obtained after hydrolysis was found to be in the following order: alcalase > chymotrypsin > trypsin/pepsin > flavourzyme. The hydrolysates obtained from alcalase (IC 50 = 0.8 mg/mL) were fractionated by sequential ultrafiltration at 10 and 3 kDa cutoff values and the most active fraction (<3 kDa) was further separated using reversed phase high-performance liquid chromatography (RP-HPLC). Peptide sequence identifications were carried out on highly potential fractions obtained from RP-HPLC by means of liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Sequencing of ACE inhibitory peptides present in the fraction 26 of RP-HPLC resulted in the identification of 3 peptide sequences (VVNE, VVTR, and VVGVD) not reported previously in the literature. Sequence identification of fractions 40 and 42 from RP-HPLC, which showed the highest ACE inhibitory activities (84.1% and 86.9%, respectively), resulted in the identification of more than 40 potential ACE inhibitory sequences. The results indicate that wild almond protein is a rich source of potential antihypertensive peptides and can be suggested for applications in functional foods and drinks with respect to hindrance and mitigation of hypertension after in vivo assessment. This study has shown the potential of wild almond proteins as good sources for producing ACE-inhibitory active peptides. According to this finding, peptides with higher ACE inhibitory activities could be released during the gastrointestinal digestion and contribute to the health- promoting activities of this natural protein source. © 2017 Institute of Food Technologists®.

Trophic transfer of trace metals: Subcellular compartmentalization in a polychaete and assimilation by a decapod crustacean

USGS Publications Warehouse

Rainbow, P.S.; Poirier, L.; Smith, B.D.; Brix, K.V.; Luoma, S.N.

2006-01-01

The chemical form of accumulated trace metal in prey is important in controlling the bioavailataility of dietary metal to a predator. This study investigated the trophic transfer of radiolabelled Ag, Cd and Zn from the polychaete worm Nereis diversicolor to the decapod crustacean Palaemonetes varians. We used 2 populations of worms with different proportions of accumulated metals in different subcellular fractions as prey, and loaded the worms with radiolabelled metals either from sediment or from solution. Accumulated radiolabelled metals were fractionated into 5 components : metal-rich granules (MRG), cellular debris, organelles, metallothionein-like proteins (MTLP), and other (heat-sensitive) proteins (HSP). Assimilation efficiencies (AE) of the metals by P. varians were measured from the 4 categories of prey (i.e. 2 populations, radiolabelled from sediment or solution). There were significant differences for each metal between the AEs from the different prey categories, confirming that origin of prey and route of uptake of accumulated trace metal will cause intraspecific differences in subsequent metal assimilation. Correlations were sought between AEs and selected fractions or combinations of fractions of metals in the prey-MRG, Trophically Available Metal (TAM = MTLP + HSP + organelles) and total protein (MTLP + HSP). TAM explained 28% of the variance in AEs for Ag, but no consistent relationships emerged between AEs and TAM or total protein when the metals were considered separately. AEs did, however, show significant positive regressions with both TAM and total protein when the 3 metals were considered together, explaining only about 21 % of the variance in each case. A significant negative relationship was observed between MRG and AE for all metals combined. The predator (P. varians) can assimilate dietary metal from a range of the fractions binding metals in the prey (N. diversicolor), with different assimilation efficiencies summated across these fractions. TAM and/or total protein may represent an approximate minimum for trophic availability but neither of these alone is a fully accurate predictor. ?? Inter-Research 2006.

Dynamics analysis of fractional order Yu-Wang system

NASA Astrophysics Data System (ADS)

Bhalekar, Sachin

2013-10-01

Fractional order version of a dynamical system introduced by Yu and Wang (Engineering, Technology & Applied Science Research, 2, (2012) 209-215) is discussed in this article. The basic dynamical properties of the system are studied. Minimum effective dimension 0.942329 for the existence of chaos in the proposed system is obtained using the analytical result. For chaos detection, we have calculated maximum Lyapunov exponents for various values of fractional order. Feedback control method is then used to control chaos in the system. Further, the system is synchronized with itself and with fractional order financial system using active control technique. Modified Adams-Bashforth-Moulton algorithm is used for numerical simulations.

Impact of biological activated carbon pre-treatment on the hydrophilic fraction of effluent organic matter for mitigating fouling in microfiltration.

PubMed

Pramanik, Biplob Kumar; Roddick, Felicity A; Fan, Linhua

2017-07-24

The hydrophilic (HPI) fraction of effluent organic matter, which has protein and carbohydrate contents, has a high propensity to foul low-pressure membranes. Biological activated carbon (BAC) filtration was examined as a pre-treatment for reducing the fouling of a microfiltration (MF) membrane (0.1 µm PVDF) by the HPI organic fraction extracted from a biologically treated secondary effluent (BTSE). Although the BAC removed less dissolved organic carbon, carbohydrate and protein from the HPI fraction than the granular activated carbon treatment which was used for comparison, it led to better improvement in permeate flux. This was shown to be due to the removal/breakdown of the HPI fraction resulting in less deposition of these organics on the membrane, many components of which are high molecular weight biopolymers (such as protein and carbohydrate molecules) through biodegradation and adsorption of those molecules on the biofilm and activated carbon. This study established the potential of BAC pre-treatment for reducing the HPI fouling of the membrane and thus improving the performance for the MF of BTSE for water reclamation.

Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis

NASA Astrophysics Data System (ADS)

Collin-Hansen, C.; Andersen, R. A.; Steinnes, E.

2003-05-01

A Cd-binding protein was isolated from the popular edible mushroom Boletus edulis, which is a hyperaccumulator of both Cd and Hg. Wild-growing samples of B. edulis were collected from soils rich in Cd. Cd radiotracer was added to the crude protein preparation obtained from ethanol precipitation of heat-treated cytosol. Proteins were then further separated in two consecutive steps; gel filtration and anion exchange chromatography. In both steps the Cd radiotracer profile showed only one distinct peak, which corresponded well with the profiles of endogenous Cd obtained by atomic absorption spectrophotometry (AAS). Concentrations of the essential elements Cu and Zn were low in the protein fractions high in Cd. N-terminal sequencing performed on the Cd-binding protein fractions revealed a protein with a novel amino acid sequence, which contained aromatic amino acids as well as proline. Both the N-terminal sequencing and spectrofluorimetric analysis with EDTA and ABD-F (4-aminosulfonyl-7-fluoro-2, 1, 3-benzoxadiazole) failed to detect cysteine in the Cd-binding fractions. These findings conclude that the novel protein does not belong to the metallothionein family. The results suggest a role for the protein in Cd transport and storage, and they are of importance in view of toxicology and food chemistry, but also for environmental protection.

Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques.

PubMed

Yamamoto, Takenori; Tamaki, Haruna; Katsuda, Chie; Nakatani, Kiwami; Terauchi, Satsuki; Terada, Hiroshi; Shinohara, Yasuo

2013-08-02

Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100. Copyright © 2013 Elsevier B.V. All rights reserved.

Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth

DOE PAGES

Giannone, Richard J.; Wurch, Louie L.; Podar, Mircea; ...

2015-06-25

The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, includingmore » membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.« less

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

PubMed Central

Li, Yihan; Wojcik, Roza; Dovichi, Norman J.

2010-01-01

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins. PMID:21030030

Effect of experimental hyperthyroidism on protein turnover in skeletal and cardiac muscle.

PubMed

Carter, W J; Van Der Weijden Benjamin, W S; Faas, F H

1980-10-01

Since experimental hyperthyroidism reduces skeletal muscle mass while simultaneously increasing cardiac muscle mass, the effect of hyperthyroidism on muscle protein degradation was compared in skeletal and cardiac muscle. Pulse-labeling studies using (3H) leucine and (14C) carboxyl labeled aspartate and glutamate were carried out. Hyperthyroidism caused a 25%-29% increase in protein breakdown in both sarcoplasmic and myofibrillar fractions of skeletal muscle. Increased muscle protein degradation may be a major factor in the development of skeletal muscle wasting and weakness in hyperthyroidism. In contrast, protein breakdown appeared to be reduced 22% in the sarcoplasmic fraction of hyperthyroid heart muscle and was unchanged in the myofibrillar fraction. Possible reasons for the contrasting effects of hyperthyroidism on skeletal and cardiac muscle include increased sensitivity of the hyperthyroid heart to catecholamines, increased cardiac work caused by the hemodynamic effects of hyperthyroidism, and a different direct effect of thyroid hormone at the nuclear level in cardiac as opposed to skeletal muscle.

Regulation of Phosphorylation of a Specific Protein in Toad-Bladder Membrane by Antidiuretic Hormone and Cyclic AMP, and Its Possible Relationship to Membrane Permeability Changes

PubMed Central

DeLorenzo, Robert J.; Walton, Kenneth G.; Curran, Peter F.; Greengard, Paul

1973-01-01

Phosphorylation of a specific protein was decreased in intact toad bladders by exposure to either antidiuretic hormone or monobutyryl cyclic AMP. The decrease in phosphorylation caused by these agents preceded the change in electrical potential difference (an indicator of the rate of sodium ion transport) observed in response to the same compounds. The addition of cyclic AMP to homogenates of toad bladder led to a decrease in phosphorylation of the same, or a similar, protein. In subcellular fractionation studies, the effect of cyclic AMP on the phosphorylation of this protein was observed in those fractions rich in membrane fragments, but not in the nuclear or cell-sap fractions. These and other results are compatible with the possibility that the regulation by vasopressin and cyclic AMP of sodium and/or water transport in toad bladder may be mediated through regulation of the phosphorylation of this specific protein. Images PMID:4351809

Molecular chaperone properties of the high molecular weight aggregate from aged lens

NASA Technical Reports Server (NTRS)

Takemoto, L.; Boyle, D.; Spooner, B. S. (Principal Investigator)

1994-01-01

The high molecular weight aggregate (HMWA) fraction was isolated from the water soluble proteins of aged bovine lenses. Its composition and ability to inhibit heat-induced denaturation and aggregation were compared with the lower molecular weight, oligomeric fraction of alpha isolated from the same lens. Although the major components of both fractions were the alpha-A and alpha-B chains, the HMWA fraction possessed a decreased ability to protect other proteins against heat-induced denaturation and aggregation. Immunoelectron microscopy of both fractions demonstrated that alpha particles from the HMWA fraction contained increased amounts of beta and gamma crystallins, bound to a central region of the supramolecular complex. Together, these results demonstrate that alpha crystallins found in the HMWA fraction possess a decreased ability to protect against heat-induced denaturation and aggregation, and suggest that at least part of this decrease could be due to the increased presence of beta and gamma crystallins complexed to the putative chaperone receptor site of the alpha particles.

Pumpkin (Cucurbita maxima) seed proteins: sequential extraction processing and fraction characterization.

PubMed

Rezig, Leila; Chibani, Farhat; Chouaibi, Moncef; Dalgalarrondo, Michèle; Hessini, Kamel; Guéguen, Jacques; Hamdi, Salem

2013-08-14

Seed proteins extracted from Tunisian pumpkin seeds ( Cucurbita maxima ) were investigated for their solubility properties and sequentially extracted according to the Osborne procedure. The solubility of pumpkin proteins from seed flour was greatly influenced by pH changes and ionic strength, with higher values in the alkaline pH regions. It also depends on the seed defatting solvent. Protein solubility was decreased by using chloroform/methanol (CM) for lipid extraction instead of pentane (P). On the basis of differential solubility fractionation and depending on the defatting method, the alkali extract (AE) was the major fraction (42.1 (P), 22.3% (CM)) compared to the salt extract (8.6 (P), 7.5% (CM)). In salt, alkali, and isopropanol extracts, all essential amino acids with the exceptions of threonine and lysine met the minimum requirements for preschool children (FAO/WHO/UNU). The denaturation temperatures were 96.6 and 93.4 °C for salt and alkali extracts, respectively. Pumpkin protein extracts with unique protein profiles and higher denaturation temperatures could impart novel characteristics when used as food ingredients.

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast.

PubMed

Zhao, Yimeng; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

2016-10-07

We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with a trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. From this, 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than that of previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified.

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Presley, Andrew D; Fuller, Kathryn M; Arriaga, Edgar A

2003-08-05

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.

Identification of Ina proteins from Fusarium acuminatum

NASA Astrophysics Data System (ADS)

Scheel, Jan Frederik; Kunert, Anna Theresa; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

2015-04-01

Freezing of water above -36° C is based on ice nucleation activity (INA) mediated by ice nucleators (IN) which can be of various origins. Beside mineral IN, biological particles are a potentially important source of atmospheric IN. The best-known biological IN are common plant-associated bacteria. The IN activity of these bacteria is induced by a surface protein on the outer cell membrane, which is fully characterized. In contrast, much less is known about the nature of fungal IN. The fungal genus Fusarium is widely spread throughout the earth. It belongs to the Ascomycota and is one of the most severe fungal pathogens. It can affect a variety of organisms from plants to animals including humans. INA of Fusarium was already described about 30 years ago and INA of Fusarium as well as other fungal genera is assumed to be mediated by proteins or at least to contain a proteinaceous compound. Although many efforts were made the precise INA machinery of Fusarium and other fungal species including the proteins and their corresponding genes remain unidentified. In this study preparations from living fungal samples of F. acuminatum were fractionated by liquid chromatography and IN active fractions were identified by freezing assays. SDS-page and de novo sequencing by mass spectrometry were used to identify the primary structure of the protein. Preliminary results show that the INA protein of F. acuminatum is contained in the early size exclusion chromatography fractions indicating a high molecular size. Moreover we could identify a single protein band from IN active fractions at 130-145 kDa corresponding to sizes of IN proteins from bacterial species. To our knowledge this is for the first time an isolation of a single protein from in vivo samples, which can be assigned as IN active from Fusarium.

On the adaptive sliding mode controller for a hyperchaotic fractional-order financial system

NASA Astrophysics Data System (ADS)

Hajipour, Ahamad; Hajipour, Mojtaba; Baleanu, Dumitru

2018-05-01

This manuscript mainly focuses on the construction, dynamic analysis and control of a new fractional-order financial system. The basic dynamical behaviors of the proposed system are studied such as the equilibrium points and their stability, Lyapunov exponents, bifurcation diagrams, phase portraits of state variables and the intervals of system parameters. It is shown that the system exhibits hyperchaotic behavior for a number of system parameters and fractional-order values. To stabilize the proposed hyperchaotic fractional system with uncertain dynamics and disturbances, an efficient adaptive sliding mode controller technique is developed. Using the proposed technique, two hyperchaotic fractional-order financial systems are also synchronized. Numerical simulations are presented to verify the successful performance of the designed controllers.

Ergodicity convergence test suggests telomere motion obeys fractional dynamics

NASA Astrophysics Data System (ADS)

Kepten, E.; Bronshtein, I.; Garini, Y.

2011-04-01

Anomalous diffusion, observed in many biological processes, is a generalized description of a wide variety of processes, all obeying the same law of mean-square displacement. Identifying the basic mechanisms of these observations is important for deducing the nature of the biophysical systems measured. We implement a previously suggested method for distinguishing between fractional Langevin dynamics, fractional Brownian motion, and continuous time random walk based on the ergodic nature of the data. We apply the method together with the recently suggested P-variation test and the displacement correlation to the lately measured dynamics of telomeres in the nucleus of mammalian cells and find strong evidence that the telomeres motion obeys fractional dynamics. The ergodic dynamics are observed experimentally to fit fractional Brownian or Langevin dynamics.

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

PubMed

Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

2017-08-01

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

Dynamics of proteins: Light scattering study of dilute and dense colloidal suspensions of eye lens homogenates

NASA Astrophysics Data System (ADS)

Giannopoulou, A.; Aletras, A. J.; Pharmakakis, N.; Papatheodorou, G. N.; Yannopoulos, S. N.

2007-11-01

We report a dynamic light scattering study on protein suspensions of bovine lens homogenates at conditions (pH and ionic strength) similar to the physiological ones. Light scattering data were collected at two temperatures, 20 and 37°C, over a wide range of concentrations from the very dilute limit up to the dense regime approaching the physiological lens concentration. A comparison with experimental data from intact bovine lenses was advanced, revealing differences between dispersions and lenses at similar concentrations. In the dilute regime, two scattering entities were detected and identified with the long-time self-diffusion modes of α-crystallins and their aggregates, which naturally exist in lens nucleus. Upon increasing protein concentration, significant changes in time correlation function were observed starting at ˜75mgml-1, where a new mode originating from collective diffusive motions becomes visible. Self-diffusion coefficients are temperature insensitive, whereas the collective diffusion coefficient depends strongly on temperature revealing a reduction of the net repulsive interparticle forces with decreasing temperature. While there are no rigorous theoretical approaches on particle diffusion properties for multicomponent, nonideal hard sphere polydispersed systems, as the suspensions studied here, a discussion of the volume fraction dependence of the long-time self-diffusion coefficient in the context of existing theoretical approaches was undertaken. This study is purported to provide some insight into the complex light scattering pattern of intact lenses and the interactions between the constituent proteins that are responsible for lens transparency. This would lead to understand basic mechanisms of specific protein interactions that lead to lens opacification (cataract) under pathological conditions.

Effect of the tocotrienol-rich fraction on the lifespan and oxidative biomarkers in Caenorhabditis elegans under oxidative stress

PubMed Central

Aan, Goon Jo; Zainudin, Mohd Shahril Aszrin; Karim, Noralisa Abdul; Ngah, Wan Zurinah Wan

2013-01-01

OBJECTIVE: This study was performed to determine the effect of the tocotrienol-rich fraction on the lifespan and oxidative status of C. elegans under oxidative stress. METHOD: Lifespan was determined by counting the number of surviving nematodes daily under a dissecting microscope after treatment with hydrogen peroxide and the tocotrienol-rich fraction. The evaluated oxidative markers included lipofuscin, which was measured using a fluorescent microscope, and protein carbonyl and 8-hydroxy-2′-deoxyguanosine, which were measured using commercially available kits. RESULTS: Hydrogen peroxide-induced oxidative stress significantly decreased the mean lifespan of C. elegans, which was restored to that of the control by the tocotrienol-rich fraction when administered before or both before and after the hydrogen peroxide. The accumulation of the age marker lipofuscin, which increased with hydrogen peroxide exposure, was decreased with upon treatment with the tocotrienol-rich fraction (p<0.05). The level of 8-hydroxy-2′-deoxyguanosine significantly increased in the hydrogen peroxide-induced group relative to the control. Treatment with the tocotrienol-rich fraction before or after hydrogen peroxide induction also increased the level of 8-hydroxy-2′-deoxyguanosine relative to the control. However, neither hydrogen peroxide nor the tocotrienol-rich fraction treatment affected the protein carbonyl content of the nematodes. CONCLUSION: The tocotrienol-rich fraction restored the lifespan of oxidative stress-induced C. elegans and reduced the accumulation of lipofuscin but did not affect protein damage. In addition, DNA oxidation was increased. PMID:23778402

Effect of the tocotrienol-rich fraction on the lifespan and oxidative biomarkers in Caenorhabditis elegans under oxidative stress.

PubMed

Aan, Goon Jo; Zainudin, Mohd Shahril Aszrin; Karim, Noralisa Abdul; Ngah, Wan Zurinah Wan

2013-05-01

This study was performed to determine the effect of the tocotrienol-rich fraction on the lifespan and oxidative status of C. elegans under oxidative stress. Lifespan was determined by counting the number of surviving nematodes daily under a dissecting microscope after treatment with hydrogen peroxide and the tocotrienol-rich fraction. The evaluated oxidative markers included lipofuscin, which was measured using a fluorescent microscope, and protein carbonyl and 8-hydroxy-2'-deoxyguanosine, which were measured using commercially available kits. Hydrogen peroxide-induced oxidative stress significantly decreased the mean lifespan of C. elegans, which was restored to that of the control by the tocotrienol-rich fraction when administered before or both before and after the hydrogen peroxide. The accumulation of the age marker lipofuscin, which increased with hydrogen peroxide exposure, was decreased with upon treatment with the tocotrienol-rich fraction (p<0.05). The level of 8-hydroxy-2'-deoxyguanosine significantly increased in the hydrogen peroxide-induced group relative to the control. Treatment with the tocotrienol-rich fraction before or after hydrogen peroxide induction also increased the level of 8-hydroxy-2'-deoxyguanosine relative to the control. However, neither hydrogen peroxide nor the tocotrienol-rich fraction treatment affected the protein carbonyl content of the nematodes. The tocotrienol-rich fraction restored the lifespan of oxidative stress-induced C. elegans and reduced the accumulation of lipofuscin but did not affect protein damage. In addition, DNA oxidation was increased.

Fractional ultrabasic-basic evolution of upper-mantle magmatism: Evidence from xenoliths in kimberlites, inclusions in diamonds and experiments

NASA Astrophysics Data System (ADS)

Litvin, Yuriy; Kuzyura, Anastasia

2017-04-01

Ultrabasic peridotites and pyroxenites together with basic eclogites are the upper-mantle in situ rocks among xenoliths in kimberlites. Occasionally their diamond-bearing varieties have revealed within the xenoliths. Therewith the compositions of rock-forming minerals demonstrate features characteristic for primary diamond-included minerals of peridotite and eclogite parageneses (the elevated contents of Cr-component in peridotitic garnets and Na-jadeitic component in eclogitic clinopyroxenes). High-pressure experimental study of melting equilibria on the multicomponent peridotie-pyroxenite system olivine Ol - orthopyroxene Opx - clinopyroxene Cpx - garnet Grt showed that Opx disappeared in the peritectic reaction Opx+L→Cpx (Litvin, 1991). As a result, the invariant peritectic equilibrium Ol+Opx+Cpx+Grt+L of the ultrabasic system was found to transform into the univariant cotectic assemblage Ol+Cpx+Grt+L. Further experimental investigation showed that olivine reacts with jadeitic component (Jd) with formation of garnet at higher 4.5 GPa (Gasparik, Litvin, 1997). Study of melting relations in the multicomponent system Ol - Cpx - Jd permits to discover the peritectic point Ol+Omph+Grt+L (where Omph - omphacitic clinopyroxene) at concentration 3-4 wt.% Jd-component in the system. The reactionary loss of Opx and Ol makes it possible to transform the 4-phase garnet lherzolite ultrabasic association into the bimineral eclogite assemblage. The regime of fractional Ol, Cpx and Grt crystallization must be accompanied by increasing content of jadeitic component in residual melts that causes the complete "garnetization of olivine". In the subsequent evolution, the melts would have to fractionate for basic SiO2-saturated compositions responsible for petrogenesis of eclogite varieties marked with accessory corundum Crn, kyanite Ky and coesite Coe. Both the peritectic mechanisms occur in regime of fractional crystallization. The sequence of the upper-mantle fractional ultrabasic-basic magmatic evolution and petrogenesis may be controlled by the following melting relations: from Ol, Opx, L field to cotectic curve Ol, Opx, Cpx, L, peritectic point Ol, Opx, Cpx, Grt, L (loss of Opx), cotectic curve Ol, (Cpx+Jd), Grt, L, peritectic point Ol, (Cpx→Omph), Grt, L (loss of Ol), divariant field Omph,Grt,L, cotectic curve Ky, Omph, Grt, L, eutectic point Ky,Coe,Omph, Grt,L, subsolidus assemblage Ky,Coe,Omph, Grt. The fractional ultrabasic-basic evolution of the upper-mantle silicate-carbonate-carbon melts-solutions, which are responsible for genesis of diamond-and-inclusions associations and diamond-bearing peridotites and eclogites, follows the similar physico-chemical mechanisms (Litvin et al., 2016). This is illustrated by fractional syngenesis diagram for diamonds and associated minerals which construction is based on evidence from high pressure experiments. References Gasparik T., Litvin Yu.A (1997). Stability of Na2Mg2Si2O7 and melting relations on the forsterite - jadeite join at pressures up to 22 GPa. Eur, J. Mineral. 9(2), 311-326. Litvin Yu.A. (1991). Physico-Chemical Study of Melting of Materials from the Deep Earth. Moscow: Nauka. 312 p. Litvin Yu.A., Spivak A.V., Kuzyura A.V. (2016). Fundamentals of the mantle-carbonatite concept of diamond genesis, Geochemistry Internat. 34(10), 839-857.

Piperazines for peptide carboxyl group derivatization: effect of derivatization reagents and properties of peptides on signal enhancement in matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Qiao, Xiaoqiang; Sun, Liangliang; Chen, Lingfan; Zhou, Yuan; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-03-15

Piperazine-based derivatives, including 1-(2-pyridyl)piperazine (2-PP), 1-(2-pyrimidyl)piperazine (2-PMP), 1-(4-pyridyl)piperazine (4-PP), and 1-(1-methyl-4-piperidinyl)piperazine (M-PP), were used for the derivatization of carboxyl groups on peptides with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt) as coupling reagents, and trifluoroacetic acid (TFA) as activator. Taking synthetic peptides RVYVHPI (RI-7) and APGDRIYVHPF (AF-11) as samples, the yields of derivatized peptides by 2-PP, 2-PMP and 4-PP were higher than 94%. The effect of piperazine derivatives on the signals of tryptic digests of α-transferrin and bovine serum albumin (BSA) was investigated, and it was found that peptides derivatized by 2-PP and 2-PMP exhibited obviously improved ionization efficiency. Furthermore, comparison of identified peptides before and after derivatization showed that peptides with low molecular weight (MW) and high pI value were preferably detected after derivatization. In addition, after derivatization with 2-PP and 2-PMP, protein myelin basic protein S, 20 kDa protein, and histone H were confidently identified from the tryptic digests of two fractions of rat brain protein separated by reversed-phase high-performance liquid chromatography (HPLC), indicating the potential application of 2-PP and 2-PMP for the highly sensitive determination of peptides in comprehensive proteome analysis. Copyright © 2011 John Wiley & Sons, Ltd.

A review of MRI evaluation of demyelination in cuprizone murine model

NASA Astrophysics Data System (ADS)

Krutenkova, E.; Pan, E.; Khodanovich, M.

2015-11-01

The cuprizone mouse model of non-autoimmune demyelination reproduces some phenomena of multiple sclerosis and is appropriate for validation and specification of a new method of non-invasive diagnostics. In the review new data which are collected using the new MRI method are compared with one or more conventional MRI tools. Also the paper reviewed the validation of MRI approaches using histological or immunohistochemical methods. Luxol fast blue histological staining and myelin basic protein immunostaining is widespread. To improve the accuracy of non-invasive conventional MRI, multimodal scanning could be applied. The new quantitative MRI method of fast mapping of the macromolecular proton fraction is a reliable biomarker of myelin in the brain and can be used for research of demyelination in animals. To date, a validation of MPF method on the CPZ mouse model of demyelination is not performed, although this method is probably the best way to evaluate demyelination using MRI.

Methods for Posttranslational Induction of Polyreactivity of Antibodies.

PubMed

Lecerf, Maxime; Jarossay, Annaelle; Kaveri, Srinivas V; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D

2017-01-01

An antibody molecule that recognizes multiple unrelated antigens is defined as polyreactive. Polyreactivity is an intrinsic characteristic of immune repertoires. Degenerated antigen binding diversifies the repertoire of specificities, thus contributing to immune defense and immune regulation. Immune repertoire contains also a fraction of immunoglobulins, which acquire polyreactivity only following contact with various protein-destabilizing or pro-oxidative substances. Posttranslational induction of the antibody polyreactivity may have important repercussion for laboratory practice, as well as in cases of pathological conditions accompanied by liberation of large quantities of pro-oxidative substances such as heme, labile iron, or reactive oxygen species. Antibodies with induced polyreactivity have been demonstrated to exert pathogen neutralization and immune regulatory potential in inflammatory conditions, suggesting that this phenomenon may be exploited for design of therapeutic strategies. In this article, we provide description of the basic procedures for uncovering of the cryptic polyreactivity of antibodies by heme, ferrous ions, and acid pH solution.

Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

PubMed Central

Lozano-Ramos, Inés; Bancu, Ioana; Oliveira-Tercero, Anna; Armengol, María Pilar; Menezes-Neto, Armando; Del Portillo, Hernando A.; Lauzurica-Valdemoros, Ricardo; Borràs, Francesc E.

2015-01-01

Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs. When analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm–Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species. To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches. PMID:26025625

A study of the influence of charged residues on β-hairpin formation by nuclear magnetic resonance and molecular dynamics.

PubMed

Makowska, Joanna; Zmudzińska, Wioletta; Uber, Dorota; Chmurzyński, Lech

2014-12-01

Chain reversals are often nucleation sites in protein folding. The β-hairpins of FBP28 WW domain and IgG are stable and have been proved to initiate the folding and are, therefore, suitable for studying the influence of charged residues on β-hairpin conformation. In this paper, we carried out NMR examination of the conformations in solution of two fragments from the FPB28 protein (PDB code: 1E0L) (N-terminal part) namely KTADGKT-NH2 (1E0L 12-18, D7) and YKTADGKTY-NH2 (1E0L 11-19, D9), one from the B3 domain of the protein G (PDB code: 1IGD), namely DDATKT-NH2 (1IGD 51-56) (Dag1), and three variants of Dag1 peptide: DVATKT-NH2 (Dag2), OVATKT-NH2 (Dag3) and KVATKT-NH2 (Dag4), respectively, in which the original charged residue were replaced with non-polar residues or modified charged residues. It was found that both the D7 and D9 peptides form a large fraction bent conformations. However, no hydrophobic contacts between the terminal Tyr residues of D9 occur, which suggests that the presence of a pair of like-charged residues stabilizes chain reversal. Conversely, only the Dag1 and Dag2 peptides exhibit some chain reversal; replacing the second aspartic-acid residue with a valine and the first one with a basic residue results in a nearly extended conformation. These results suggest that basic residues farther away in sequence can result in stabilization of chain reversal owing to screening of the non-polar core. Conversely, smaller distance in sequence prohibits this screening, while the presence oppositely-charged residues can stabilize a turn because of salt-bridge formation.

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

PubMed

Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

2015-10-01

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis. Copyright © 2015 John Wiley & Sons, Ltd.

Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

PubMed

Ruiz-Baca, Estela; Villagómez-Castro, Julio C; Leal-Morales, Carlos A; Sabanero-López, Myrna; Flores-Carreón, Arturo; López-Romero, Everardo

2005-01-01

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Fractions division knowledge of elementary school student: The case of Lala

NASA Astrophysics Data System (ADS)

Purnomo, Yoppy Wahyu; Widowati, Chairunnisa; Aziz, Tian Abdul; Pramudiani, Puri

2017-08-01

Division of fractions is often acknowledged by mysterious rule which is not based on conceptual knowledge. The purpose of the study was to explore elementary school student's knowledge of division fractions. For this purpose, a case study was conducted. The participant of the study was Lala (pseudonym) who enrolled at one elementary school in East Jakarta. The data were collected by administering written test and semi-structured interview respectively. The findings of the study indicated that Lala was able to describe strategy of division fractions as inverse of repeated addition flexibly. She also had basic understanding of fractions division concept as equal sharing, but when she was challenged with advance problems, she performed poorly. Lala also encountered difficulty when dealing with dividing fraction by fraction problem in which she interpreted it as subtraction problem. In this case, her procedural knowledge was likely to be more salient than her conceptual knowledge.

Implementation of quantum and classical discrete fractional Fourier transforms.

PubMed

Weimann, Steffen; Perez-Leija, Armando; Lebugle, Maxime; Keil, Robert; Tichy, Malte; Gräfe, Markus; Heilmann, René; Nolte, Stefan; Moya-Cessa, Hector; Weihs, Gregor; Christodoulides, Demetrios N; Szameit, Alexander

2016-03-23

Fourier transforms, integer and fractional, are ubiquitous mathematical tools in basic and applied science. Certainly, since the ordinary Fourier transform is merely a particular case of a continuous set of fractional Fourier domains, every property and application of the ordinary Fourier transform becomes a special case of the fractional Fourier transform. Despite the great practical importance of the discrete Fourier transform, implementation of fractional orders of the corresponding discrete operation has been elusive. Here we report classical and quantum optical realizations of the discrete fractional Fourier transform. In the context of classical optics, we implement discrete fractional Fourier transforms of exemplary wave functions and experimentally demonstrate the shift theorem. Moreover, we apply this approach in the quantum realm to Fourier transform separable and path-entangled biphoton wave functions. The proposed approach is versatile and could find applications in various fields where Fourier transforms are essential tools.

Implementation of quantum and classical discrete fractional Fourier transforms

PubMed Central

Weimann, Steffen; Perez-Leija, Armando; Lebugle, Maxime; Keil, Robert; Tichy, Malte; Gräfe, Markus; Heilmann, René; Nolte, Stefan; Moya-Cessa, Hector; Weihs, Gregor; Christodoulides, Demetrios N.; Szameit, Alexander

2016-01-01

Fourier transforms, integer and fractional, are ubiquitous mathematical tools in basic and applied science. Certainly, since the ordinary Fourier transform is merely a particular case of a continuous set of fractional Fourier domains, every property and application of the ordinary Fourier transform becomes a special case of the fractional Fourier transform. Despite the great practical importance of the discrete Fourier transform, implementation of fractional orders of the corresponding discrete operation has been elusive. Here we report classical and quantum optical realizations of the discrete fractional Fourier transform. In the context of classical optics, we implement discrete fractional Fourier transforms of exemplary wave functions and experimentally demonstrate the shift theorem. Moreover, we apply this approach in the quantum realm to Fourier transform separable and path-entangled biphoton wave functions. The proposed approach is versatile and could find applications in various fields where Fourier transforms are essential tools. PMID:27006089

CRYSTALLINE PNEUMOCOCCUS ANTIBODY

PubMed Central

Northrop, John H.; Goebel, Walther F.

1949-01-01

1. The immune precipitate formed by antipneumococcus horse serum and the specific polysaccharide is not hydrolyzed by trypsin as is the diphtheria toxin-antitoxin complex, and purified pneumococcus antibody cannot be isolated by the method used for the isolation and crystallization of diphtheria antitoxin. 2. Type I pneumococcus antibody, completely precipitable by Type I polysaccharide, may be obtained from immune horse serum globulin by precipitation of the inert proteins with acid potassium phthalate. 3. The antibody obtained in this way may be fractionated by precipitation with ammonium sulfate into three main parts. One is insoluble in neutral salts but soluble from pH 4.5 to 3.0 and from pH 9.5 to 10.5. This is the largest fraction. A second fraction is soluble in 0.05 to 0.2 saturated ammonium sulfate and the third fraction is soluble in 0.2 saturated ammonium sulfate and precipitated by 0.35 saturated ammonium sulfate. The second fraction can be further separated by precipitation with 0.17 saturated ammonium sulfate to yield a small amount of protein which is soluble in 0.17 saturated ammonium sulfate but insoluble in 0.25 saturated ammonium sulfate. This fraction crystallizes in poorly formed, rounded rosettes. 4. The crystallization does not improve the purity of the antibody and is accompanied by the formation of an insoluble protein as in the case of diphtheria antitoxin. 5. None of the fractions obtained is even approximately homogeneous as determined by solubility measurements. 6. Purified antibody has also been obtained by dissociating the antigen-antibody complex. 7. The protective value of the fractions is quite different; that of the dissociated antibody being the highest and that of the insoluble fraction, the lowest. 8. All the fractions are immunologically specific since they do not precipitate with Type II polysaccharide nor protect against Type II pneumococci. 9. All the fractions give a positive precipitin reaction with antihorse rabbit serum. The dissociated antibody gives the least reaction. 10. Comparison of the various fractions, either by their solubility in salt solution or through immunological reactions, indicates that there are a large number of proteins present in immune horse serum, all of which precipitate with the specific polysaccharide but which have very different protective values, different reactions with antihorse rabbit serum, and different solubility in salt solutions. PMID:18131872

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane.

PubMed

Rabani, Vahideh; Montange, Damien; Meneveau, Nicolas; Davani, Siamak

2017-10-11

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

Binding of vitamin A by casein micelles in commercial skim milk

PubMed Central

Mohan, M. S.; Jurat-Fuentes, J. L.; Harte, F.

2015-01-01

Recent studies have shown that reassembled micelles formed by caseinates and purified casein fractions (αs- and β-casein) bind to hydrophobic compounds, including curcumin, docosahexaenoic acid, and vitamin D. However, limited research has been done on the binding of hydrophobic compounds by unmodified casein micelles in skim milk. In the present study, we investigated the ability of casein micelles in commercial skim milk to associate with vitamin A (retinyl palmitate), a fat-soluble vitamin commonly used to fortify milk. Milk protein fractions from different commercially available skim milk samples subjected to different processing treatments, including pasteurized, ultrapasteurized, organic pasteurized, and organic ultrapasteurized milks, were separated by fast protein liquid chromatography. The fractions within each peak were combined and freeze-dried. Sodium dodecyl sulfate-PAGE with silver staining was used to identify the proteins present in each of the fractions. The skim milk samples and fractions were extracted for retinyl palmitate and quantified against a standard using normal phase-HPLC. Retinyl palmitate was found to associate with the fraction of skim milk containing caseins, whereas the other proteins (BSA, β-lactoglobulin, α-lactalbumin) did not show any binding. The retinyl palmitate content in the various samples ranged from 1.59 to 2.48 μg of retinyl palmitate per mL of milk. The casein fractions contained between 14 and 40% of total retinyl palmitate in the various milks tested. The variation in the retention of vitamin A by caseins was probably explained by differences in the processing of different milk samples, including thermal treatment, the form of vitamin A emulsion used for fortification, and the point of fortification during processing. Unmodified casein micelles have a strong intrinsic affinity toward the binding of vitamin A used to fortify commercially available skim milks. PMID:23261375

A sequence of basic residues in the porcine circovirus type 2 capsid protein is crucial for its co-expression and co-localization with the replication protein.

PubMed

Huang, Liping; Van Renne, Nicolaas; Liu, Changming; Nauwynck, Hans J

2015-12-01

Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep) and the capsid protein (Cap). Cap displays a conserved stretch of basic residues situated on the inside of the capsid, whose role is so far unknown. We used a reverse-genetics approach to investigate its function and found that mutations in these amino acids hindered Cap mRNA translation and hampered Cap/Rep co-localization, yielding unfit viruses. Intriguingly, co-transfection with a WT PCV2 of a different genotype partially rescued mutant Cap expression, showing the importance of this basic pattern for efficient translation of Cap mRNA into protein. Our results show that Cap and Rep are expressed independently of each other, and that this amino acid sequence of Cap is vital for virus propagation. This study provides a method for studying unfit PCV2 virions and offers new insights into the intracellular modus vivendi of PCV2.

Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages.

PubMed

Beatty, W L; Russell, D G

2000-12-01

Considerable effort has focused on the identification of proteins secreted from Mycobacterium spp. that contribute to the development of protective immunity. Little is known, however, about the release of mycobacterial proteins from the bacterial phagosome and the potential role of these molecules in chronically infected macrophages. In the present study, the release of mycobacterial surface proteins from the bacterial phagosome into subcellular compartments of infected macrophages was analyzed. Mycobacterium bovis BCG was surface labeled with fluorescein-tagged succinimidyl ester, an amine-reactive probe. The fluorescein tag was then used as a marker for the release of bacterial proteins in infected macrophages. Fractionation studies revealed bacterial proteins within subcellular compartments distinct from mycobacteria and mycobacterial phagosomes. To identify these proteins, subcellular fractions free of bacteria were probed with mycobacterium-specific antibodies. The fibronectin attachment protein and proteins of the antigen 85-kDa complex were identified among the mycobacterial proteins released from the bacterial phagosome.

Protein precipitation behavior of condensed tannins from Lotus pedunculatus and Trifolium repens with different mean degrees of polymerization

USDA-ARS?s Scientific Manuscript database

The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited...

Studies on a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis. 1 isolation and characterization.

PubMed

Solov'eva, T F; Yermak, I M; Bondarenko, O D; Frolova, G M; Ovodov, Y S

1979-01-01

A comparative study of various procedures of a lipopolysaccharide-protein complex (LPPC) from Yersinia pseudotuberculosis was carried out. The materials obtained were fractionated by molecular-sieve chromatography on Sepharose 2B resulting in highly aggregated complexes with antigen activity. LPPC aggregates dissociated in the presence of sodium dodecylsulphate (SDS) and urea. The chemical composition and serologic properties of fractions obtained are under consideration. The protein component of the complex consists of two major polypeptides (molecular weights--45,000 and 20,000) and some minor ones. The LPS component appeared to give 2--3 narrow bands in gel under conditions of SDS-polyacrylamide gel electrophoresis. It is suggested that such fractionation is caused by LPS association-dissociation in the course of electrophoresis.

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

PubMed Central

Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

1990-01-01

We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis. Images PMID:2106493

Non-standard finite difference and Chebyshev collocation methods for solving fractional diffusion equation

NASA Astrophysics Data System (ADS)

Agarwal, P.; El-Sayed, A. A.

2018-06-01

In this paper, a new numerical technique for solving the fractional order diffusion equation is introduced. This technique basically depends on the Non-Standard finite difference method (NSFD) and Chebyshev collocation method, where the fractional derivatives are described in terms of the Caputo sense. The Chebyshev collocation method with the (NSFD) method is used to convert the problem into a system of algebraic equations. These equations solved numerically using Newton's iteration method. The applicability, reliability, and efficiency of the presented technique are demonstrated through some given numerical examples.

Nucleolar Trafficking of Nucleostemin Family Proteins: Common versus Protein-Specific Mechanisms▿ §

PubMed Central

Meng, Lingjun; Zhu, Qubo; Tsai, Robert Y. L.

2007-01-01

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins. PMID:17923687

On the function of chitin synthase extracellular domains in biomineralization.

PubMed

Weiss, Ingrid M; Lüke, Florian; Eichner, Norbert; Guth, Christina; Clausen-Schaumann, Hauke

2013-08-01

Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface. Copyright © 2013 Elsevier Inc. All rights reserved.

A Community Standard Format for the Representation of Protein Affinity Reagents*

PubMed Central

Gloriam, David E.; Orchard, Sandra; Bertinetti, Daniela; Björling, Erik; Bongcam-Rudloff, Erik; Borrebaeck, Carl A. K.; Bourbeillon, Julie; Bradbury, Andrew R. M.; de Daruvar, Antoine; Dübel, Stefan; Frank, Ronald; Gibson, Toby J.; Gold, Larry; Haslam, Niall; Herberg, Friedrich W.; Hiltke, Tara; Hoheisel, Jörg D.; Kerrien, Samuel; Koegl, Manfred; Konthur, Zoltán; Korn, Bernhard; Landegren, Ulf; Montecchi-Palazzi, Luisa; Palcy, Sandrine; Rodriguez, Henry; Schweinsberg, Sonja; Sievert, Volker; Stoevesandt, Oda; Taussig, Michael J.; Ueffing, Marius; Uhlén, Mathias; van der Maarel, Silvère; Wingren, Christer; Woollard, Peter; Sherman, David J.; Hermjakob, Henning

2010-01-01

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. PMID:19674966

Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwieterman, W.; Sorrentino, D.; Potter, B.J.

1988-01-01

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (V/sub O/) of (/sup 3/H)-oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound (/sup 3/H)oleate in the medium. V/sub O/ reached a maximum as the concentration of unbound oleate was increased and was significantly inhibited both by phloretin and by prior incubation ofmore » the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 ..mu..g/ml V/sub max/ was reduced from 2480 /plus minus/ 160 to 1870 /plus minus/ 80 pmol/min per 5 /times/ 10/sup 4/ adipocytes, with no change in K/sub m/. A basic kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.« less

Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

PubMed

Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

2015-05-01

High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibitory properties of bambara groundnut protein hydrolysate and peptide fractions against angiotensin-converting enzymes, renin and free radicals.

PubMed

Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Malomo, Sunday A; Aluko, Rotimi E; Amonsou, Eric O

2017-07-01

An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities. Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTS •+ to a three-fold greater extent compared to the isolate. BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

[Dynamics of amino acid and protein metabolism of laying hens after administration of 15N-labeled wheat protein. 5. Incorporation of 15N into the blood fraction and its amino acids].

PubMed

Gruhn, K; Zander, R; Kirchner, E

1987-09-01

12 colostomized laying hens which received 15N labelled wheat over 4 days were butchered 12 h, 36 h, 60 h and 108 h (3 animals each) after the last 15N application. The intake of 15N excess (15N') from the wheat amounted to 540 mg 15N' during the application period. The 15N' in the blood plasma decreased after the last 15N' application from 0.76 atom-% to 0.55 atom-% after 108 h, the labelling of the corpuscular components at the same measuring points increased from 0.28 to 0.50 atom-% 15N'. 96.6% of the plasma 15N' and 93.8% of that in the corpuscles is precipitable in trichloric acetic acid. The atom-% 15N' of histidine in the total blood remained unchanged in dependence on the butchering time. The 15N amount in lysine and arginine and that in the non-basic amino acids decreased inconsiderably in the period between 12 h and 108 h after the last 15N' wheat feeding.

Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom.

PubMed

Olmedo, Hidekel; Herrera, María; Rojas, Leonardo; Villalta, Mauren; Vargas, Mariángela; Leiguez, Elbio; Teixeira, Catarina; Estrada, Ricardo; Gutiérrez, José María; León, Guillermo; Montero, Mavis L

2014-01-01

The adjuvanticity of aluminum hydroxide and calcium phosphate on the antibody response in mice towards the venom of the snake Bothrops asper was studied. It was found that, in vitro, most of the venom proteins are similarly adsorbed by both mineral salts, with the exception of some basic phospholipases A2, which are better adsorbed by calcium phosphate. After injection, the adjuvants promoted a slow release of the venom, as judged by the lack of acute toxicity when lethal doses of venom were administered to mice. Leukocyte recruitment induced by the venom was enhanced when it was adsorbed on both mineral salts; however, venom adsorbed on calcium phosphate induced a higher antibody response towards all tested HPLC fractions of the venom. On the other hand, co-precipitation of venom with calcium phosphate was the best strategy for increasing: (1) the capacity of the salt to couple venom proteins in vitro; (2) the venom ability to induce leukocyte recruitment; (3) phagocytosis by macrophages; and (4) a host antibody response. These findings suggest that the chemical nature is not the only one determining factor of the adjuvant activity of mineral salts.

Purification of a crystallin domain of Yersinia crystallin from inclusion bodies and its comparison to native protein from the soluble fraction.

PubMed

Jobby, M K; Sharma, Yogendra

2006-09-01

It has been established that many heterologously produced proteins in E. coli accumulate as insoluble inclusion bodies. Methods for protein recovery from inclusion bodies involve solubilization using chemical denaturants such as urea and guanidine hydrochloride, followed by removal of denaturant from the solution to allow the protein to refold. In this work, we applied on-column refolding and purification to the second crystallin domain D2 of Yersinia crystallin isolated from inclusion bodies. We also purified the protein from the soluble fraction (without using any denaturant) to compare the biophysical properties and conformation, although the yield was poor. On-column refolding method allows rapid removal of denaturant and refolding at high protein concentration, which is a limitation in traditionally used methods of dialysis or dilution. We were also able to develop methods to remove the co-eluting nucleic acids during chromatography from the protein preparation. Using this protocol, we were able to rapidly refold and purify the crystallin domain using a two-step process with high yield. We used biophysical techniques to compare the conformation and calcium-binding properties of the protein isolated from the soluble fraction and inclusion bodies. Copyright 2006 John Wiley & Sons, Ltd.

Amino acid sequences of peptides from a chymotryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

PubMed Central

Corfield, M. C.; Fletcher, J. C.

1969-01-01

1. A chymotryptic digest of the protein fraction U.S.3. from oxidized wool was separated into 51 peptide fractions by chromatography on a column of cation-exchange resin. 2. The less acidic fractions were separated into their component peptides by a combination of cation-exchange-resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid sequences of 34 of these peptides were elucidated, and those of 14 others partially determined. 4. Overlaps between the tryptic and chymotryptic peptides from fraction U.S.3 have enabled ten extended amino acid sequences to be deduced, the longest containing 20 amino acid residues. 5. The relevance of the results to the structures of the helical and non-helical regions of wool is discussed. PMID:5395876

Methods for validating the presence of and characterizing proteins deposited onto an array

DOEpatents

Schabacker, Daniel S.

2010-09-21

A method of determining if proteins have been transferred from liquid-phase protein fractions to an array comprising staining the array with a total protein stain and imaging the array, optionally comparing the staining with a standard curve generated by staining known amounts of a known protein on the same or a similar array; a method of characterizing proteins transferred from liquid-phase protein fractions to an array including staining the array with a post-translational modification-specific (PTM-specific) stain and imaging the array and, optionally, after staining the array with a PTM-specific stain and imaging the array, washing the array, re-staining the array with a total protein stain, imaging the array, and comparing the imaging with the PTM-specific stain with the imaging with the total protein stain; stained arrays; and images of stained arrays.

Adiponectin is partially associated with exosomes in mouse serum.

PubMed

Phoonsawat, Worrawalan; Aoki-Yoshida, Ayako; Tsuruta, Takeshi; Sonoyama, Kei

2014-06-06

Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

PubMed Central

Cao, Zheng; Bowie, James U

2014-01-01

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α-helices compared to β-sheets by ∼0.2 kcal/mol. [2] Charge-stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ∼2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies. PMID:24501090

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line.

PubMed

Hirako, Yoshiaki; Yonemoto, Yuki; Yamauchi, Tomoe; Nishizawa, Yuji; Kawamoto, Yoshiyuki; Owaribe, Katsushi

2014-06-10

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

An insight into the antibiofilm properties of Costa Rican stingless bee honeys.

PubMed

Zamora, L G; Beukelman, C J; van den Berg, A J J; Aerts, P C; Quarles van Ufford, H C; Nijland, R; Arias, M L

2017-04-02

There is an increasing search for antibiofilm agents that either have specific activity against biofilms or may act in synergy with antimicrobials. Our objective is to examine the the antibiofilm properties of stingless bee honeys. Meliponini honeys from Costa Rica were examined along with Medihoney as a reference. All honeys were submitted to a screening composed of minimum inhibitory concentration, inhibition of biofilm formation and biofilm destruction microplate-based assays against a Staphylococcus aureus biofilm forming strain. Dialysis led to the isolation of an antibiofilm fraction in Tetragonisca angustula honeys. The honey antibiofilm fraction was evaluated for protease activity and for any synergistic effect with antibiotics on a Staphylococcus aureus biofilm. The active fraction was then separated through activity guided isolation techniques involving SDS-PAGEs, anion exchange and size exclusion fast protein liquid chromatographies. The fractions obtained and the isolated antibiofilm constituents were tested for amylase and DNase activity. A total of 57 Meliponini honeys from Costa Rica were studied in this research. The honeys studied belonged to the Tetragonisca angustula (n=36) and Melipona beecheii (n=21) species. Costa Rican Tetragonisca angustula honeys can inhibit the planktonic growth, biofilm formation, and are capable of destroying a Staphylococcus aureus biofilm. The antibiofilm effect was observed in the protein fraction of Tetragonisca angustula honeys. The biofilm destruction proteins allowed ampicillin and vancomycin to recover their antimicrobial activity over a Staphylococcus aureus biofilm. The antibiofilm proteins are of bee origin, and their activity was not due to serine, cysteine or metalloproteases. There were 2 proteins causing the antibiofilm action; these were named the Tetragonisca angustula biofilm destruction factors (TABDFs). TABDF-1 is a monomeric protein of approximately 50kDa that is responsible of the amylase activity of Tetragonisca angustula honeys. TABDF-2 is a protein monomer of approximately 75kDa. Tetragonisca angustula honeys from Costa Rica are a promising candidate for research and development of novel wound dressings focused on the treatment of acute and chronic Staphylococcus aureus biofilm wound infections.

Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

PubMed Central

Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

2013-01-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

PubMed

Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I

2013-12-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.

Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production.

PubMed

Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A

2010-10-13

Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels.

PubMed

Bikker, Paul; van Krimpen, Marinus M; van Wikselaar, Piet; Houweling-Tan, Bwee; Scaccia, Nazareno; van Hal, Jaap W; Huijgen, Wouter J J; Cone, John W; López-Contreras, Ana M

2016-01-01

The growing world population demands an increase in animal protein production. Seaweed may be a valuable source of protein for animal feed. However, a biorefinery approach aimed at cascading valorisation of both protein and non-protein seaweed constituents is required to realise an economically feasible value chain. In this study, such a biorefinery approach is presented for the green seaweed Ulva lactuca containing 225 g protein ( N  × 4.6) kg -1 dry matter (DM). The sugars in the biomass were solubilised by hot water treatment followed by enzymatic hydrolysis and centrifugation resulting in a sugar-rich hydrolysate (38.8 g L -1 sugars) containing glucose, rhamnose and xylose, and a protein-enriched (343 g kg -1 in DM) extracted fraction. This extracted fraction was characterised for use in animal feed, as compared to U. lactuca biomass. Based on the content of essential amino acids and the in vitro N (85 %) and organic matter (90 %) digestibility, the extracted fraction seems a promising protein source in diets for monogastric animals with improved characteristics as compared to the intact U. lactuca . The gas production test indicated a moderate rumen fermentation of U. lactuca and the extracted fraction, about similar to that of alfalfa. Reduction of the high content of minerals and trace elements may be required to allow a high inclusion level of U. lactuca products in animal diets. The hydrolysate was used successfully for the production of acetone, butanol, ethanol and 1,2-propanediol by clostridial fermentation, and the rhamnose fermentation pattern was studied.

Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins

PubMed Central

Chaplin, David D.; Wedner, H. James; Parker, Charles W.

1979-01-01

Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of 32P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem. 250, 4007–4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of 32P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t½=5–12s at 25°C. Between 40 and 70% of the 32P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:228657

Characterization of associated proteins and phospholipids in natural rubber latex.

PubMed

Sansatsadeekul, Jitlada; Sakdapipanich, Jitladda; Rojruthai, Porntip

2011-06-01

Non-rubber components present in natural rubber (NR) latex, such as proteins and phospholipids, are presumed to be distributed in the serum fraction as well as surrounding the rubber particle surface. The phospholipid-protein layers covering the rubber particle surface are especially interesting due to their ability to enhance the colloidal stability of NR latex. In this study, we have characterized the components surrounding the NR particle surface and investigated their role in the colloidal stability of NR particles. Proteins from the cream fraction were proteolytically removed from the NR latex and compare to those from the serum fractions using SDS-polyacrylamide gel electrophoresis revealing that both fractions contained similar proteins in certain molecular weights such as 14.5, 25 and 27 kDa. Phospholipids removed from latex by treatment with NaOH were analyzed using (1)H-NMR spectroscopy and several major signals were assignable to -(CH(2))(n)-, -CH(2)OP, -CH(2)OC═O and -OCH(2)CH(2)NH-. These signals are important evidence that indicates phospholipids associate with the rubber chain. The colloidal behavior of rubber lattices before and after removal of protein-lipid membrane was evaluated by zeta potential analysis and scanning electron microscope (SEM). The lowest zeta potential value of NR particles was observed at pH 10, consequently leading to the highest stability of rubber particles. Additionally, SEM micrographs clearly displayed a gray ring near the particle surface corresponding to the protein-lipid membrane layer. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Determination of total antioxidant capacity of milk by CUPRAC and ABTS methods with separate characterisation of milk protein fractions.

PubMed

Çekiç, Sema Demirci; Demir, Aslı; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

2015-05-01

Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant 'negative-biased' deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

PubMed

Araújo, Danielle Silva; de Sousa Lima, Patrícia; Baeza, Lilian Cristiane; Parente, Ana Flávia Alves; Melo Bailão, Alexandre; Borges, Clayton Luiz; de Almeida Soares, Célia Maria

2017-11-01

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MS E , was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture. Copyright © 2017 Elsevier B.V. All rights reserved.

Short communication: Genetic parameters for milk protein composition predicted using mid-infrared spectroscopy in the French Montbéliarde, Normande, and Holstein dairy cattle breeds.

PubMed

Sanchez, M P; Ferrand, M; Gelé, M; Pourchet, D; Miranda, G; Martin, P; Brochard, M; Boichard, D

2017-08-01

Genetic parameters for the major milk proteins were estimated in the 3 main French dairy cattle breeds (i.e. Montbéliarde, Normande, and Holstein) as part of the PhénoFinlait program. The 6 major milk protein contents as well as the total protein content (PC) were estimated from mid-infrared spectrometry on 133,592 test-day milk samples from 20,434 cows in first lactation. Lactation means, expressed as a percentage of milk (protein contents) or of protein (protein fractions), were analyzed with an animal mixed model including fixed environmental effects (herd, year × month of calving, and spectrometer) and a random genetic effect. Genetic parameter estimates were very consistent across breeds. Heritability estimates (h 2 ) were generally higher for protein fractions than for protein contents. They were moderate to high for α S1 -casein, α S2 -casein, β-casein, κ-casein, and α-lactalbumin (0.25 < h 2 < 0.72). In each breed, β-lactoglobulin was the most heritable trait (0.61 < h 2 < 0.86). Genetic correlations (r g ) varied depending on how the percentage was expressed. The PC was strongly positively correlated with protein contents but almost genetically independent from protein fractions. Protein fractions were generally in opposition, except between κ-casein and α-lactalbumin (0.39 < r g < 0.46) and κ-casein and α S2 -casein (0.36 < r g < 0.49). Between protein contents, r g estimates were positive, with highest values found between caseins (0.83 < r g < 0.98). In the 3 breeds, β-lactoglobulin was negatively correlated with caseins (-0.75 < r g < -0.08), in particular with κ-casein (-0.75 < r g < -0.55). These results, obtained from a large panel of cows of the 3 main French dairy cattle breeds, show that routinely collected mid-infrared spectra could be used to modify milk protein composition by selection. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis.

PubMed

Salamatmanesh, Mina; McCudden, Christopher R; McCurdy, Arleigh; Booth, Ronald A

2018-01-01

The International Myeloma Working Group recommendations for monitoring disease progression or response include quantitation of the involved monoclonal immunoglobulin. They have defined the minimum change criteria of ≧25% with an absolute change of no <5g/L for either minimal response or progression. Limited evidence is available to accurately determine the magnitude of change in a monoclonal protein to reflect a true change in clinical status. Here we determined the analytical and biological variability of monoclonal proteins in stable monoclonal gammopathy of undetermined significance (MGUS) patients. Analytical variability (CVa) of normal protein fractions and monoclonal proteins were assessed agarose gel-based serum protein electrophoresis. Sixteen clinically stable MGUS patients were identified from our clinical hematology database. Individual biological variability (CVi) was determined and used to calculate a monoclonal protein reference change value (RCV). Analytical variability of the normal protein fractions (albumin, alpha-1, alpha-2, beta, total gamma) ranged from 1.3% for albumin to 5.8% for the alpha-1 globulins. CVa of low (5.6g/L) and high (32.2g/L) concentration monoclonal proteins were 3.1% and 22.2%, respectively. Individual CVi of stable patients ranged from 3.5% to 24.5% with a CVi of 12.9%. The reference change value (RCV) at a 95% probability was determined to be 36.7% (low) 39.6% (high) using our CVa and CVi. Serial monitoring of monoclonal protein concentration is important for MGUS and multiple myeloma patients. Accurate criteria for interpreting a change in monoclonal protein concentration are required for appropriate decision making. We used QC results and real-world conditions to assess imprecision of serum protein fractions including low and high monoclonal protein fractions and clinically stable MGUS patients to determine CVi and RCV. The calculated RCVs of 36.7% (low) and 39.6% (high) in this study were greater that reported previously and greater than the established criteria for relapse. Response criteria may be reassessed to increase sensitivity and specificity for detection of response. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Influence of micellar calcium and phosphorus on rennet coagulation properties of cows milk.

PubMed

Malacarne, Massimo; Franceschi, Piero; Formaggioni, Paolo; Sandri, Sandro; Mariani, Primo; Summer, Andrea

2014-05-01

The main requirement for milk processed in most cheese typologies is its rennet coagulation ability. Despite the increasing number of studies, the causes for abnormal coagulation of milk are not fully understood. The aim of this study was to ascertain relationships between milk characteristics and its rennet coagulation ability, focusing on the influence of calcium (Ca) and phosphorus (P). Ca and P are essential constituents of the micelles. Micellar P can be present as part of colloidal calcium phosphate (inorganic-P) or covalently bound to caseins as phosphate groups (casein-P). Eighty one herd milk samples (SCC<400 000 cell/ml) were classified as Optimal (8), Suboptimal (39) Poor (29) and Non-coagulating milk (5), according to their rennet coagulation parameters as assessed by lactodynamographic test. Samples were analysed for their chemical composition (basic composition, protein fractions, minerals and salt equilibria), physicochemical parameters (pH and titratable acidity) and rheological properties. Optimal milk was characterised by the highest contents of major constituents, protein fractions and minerals, lowest content of chloride and highest values of titratable acidity. Non-coagulating milk was characterised by the highest values of pH and the lowest of titratable acidity. At micellar level, Optimal milk showed the highest values of colloidal Ca, casein-P and colloidal Mg (g/100 g casein), while Non-coagulating milk showed the lowest values. Interestingly, there was no statistical difference regarding the content of colloidal inorganic-P (g/100 g casein) between Optimal and Non-coagulating milks. Overall, high mineralisation of the micelle (expressed as g inorganic-P/100 g casein) positively affect its rennetability. However, excessive mineralisation could lead to a reduction of the phosphate groups (g casein-P/100 g casein) available for curd formation.

Delaware Longitudinal Study of Fraction Learning: Implications for Helping Children With Mathematics Difficulties.

PubMed

Jordan, Nancy C; Resnick, Ilyse; Rodrigues, Jessica; Hansen, Nicole; Dyson, Nancy

The goal of the present article is to synthesize findings to date from the Delaware Longitudinal Study of Fraction Learning. The study followed a large cohort of children ( N = 536) between Grades 3 and 6. The findings showed that many students, especially those with diagnosed learning disabilities, made minimal growth in fraction knowledge and that some showed only a basic grasp of the meaning of a fraction even after several years of instruction. Children with low growth in fraction knowledge during the intermediate grades were much more likely to fail to meet state standards on a broad mathematics measure at the end of Grade 6. Although a range of general and mathematics-specific competencies predicted fraction outcomes, the ability to estimate numerical magnitudes on a number line was a uniquely important marker of fraction success. Many children with mathematics difficulties have deep-seated problems related to whole number magnitude representations that are complicated by the introduction of fractions into the curriculum. Implications for helping students with mathematics difficulties are discussed.

Efficacy of an MFGM-enriched complementary food in diarrhea, anemia, and micronutrient status in infants.

PubMed

Zavaleta, Nelly; Kvistgaard, Anne Staudt; Graverholt, Gitte; Respicio, Graciela; Guija, Henry; Valencia, Norma; Lönnerdal, Bo

2011-11-01

The aim of the present study was to evaluate the efficacy of a milkfat globule membrane (MFGM)-enriched protein fraction in a complementary food, on diarrhea, anemia, and micronutrient status. A randomized, double-blind controlled design to study 550 infants, 6 to 11 months old, who received daily for 6 months a complementary food (40 g/day) with the protein source being either the MFGM protein fraction or skim milk proteins (control). Health and nutritional status of infants were examined monthly in the outpatient clinic; product intake, food patterns, and diarrhea morbidity were assessed by home visits twice per week. Hemoglobin and micronutrient status were measured at 0 and 6 months of intervention. Results are presented as the entire group and as 6 to 8 and 9 to 11 months subgroups. A total of 499 infants completed the study. Global prevalence of diarrhea was 3.84% and 4.37% in the MFGM group and control group, respectively (P < 0.05). Consumption of the MFGM protein fraction reduced episodes of bloody diarrhea (odds ratio 0.54; 95% confidence interval 0.31-0.93, P = 0.025) adjusting for anemia and potable water facilities as covariates. There were no differences between groups in anemia, serum ferritin, zinc, or folate. Addition of an MFGM-enriched protein fraction to complementary food had beneficial effects on diarrhea in infants and may thus help to improve the health of vulnerable populations.

Diazo compounds for the bioreversible esterification of proteins† †Electronic supplementary information (ESI) available: Experimental procedures, analytical data, and spectral data for novel compounds. See DOI: 10.1039/c4sc01768d Click here for additional data file.

PubMed Central

McGrath, Nicholas A.; Andersen, Kristen A.; Davis, Amy K. F.; Lomax, Jo E.

2015-01-01

A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins. PMID:25544883

Exosome and Microvesicle-Enriched Fractions Isolated from Mesenchymal Stem Cells by Gradient Separation Showed Different Molecular Signatures and Functions on Renal Tubular Epithelial Cells.

PubMed

Collino, Federica; Pomatto, Margherita; Bruno, Stefania; Lindoso, Rafael Soares; Tapparo, Marta; Sicheng, Wen; Quesenberry, Peter; Camussi, Giovanni

2017-04-01

Several studies have suggested that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) may mediate MSC paracrine action on kidney regeneration. This activity has been, at least in part, ascribed to the transfer of proteins/transcription factors and different RNA species. Information on the RNA/protein content of different MSC EV subpopulations and the correlation with their biological activity is currently incomplete. The aim of this study was to evaluate the molecular composition and the functional properties on renal target cells of MSC EV sub-populations separated by gradient floatation. The results demonstrated heterogeneity in quantity and composition of MSC EVs. Two peaks of diameter were observed (90-110 and 170-190 nm). The distribution of exosomal markers and miRNAs evaluated in the twelve gradient fractions showed an enrichment in fractions with a flotation density of 1.08-1.14 g/mL. Based on this observation, we evaluated the biological activity on renal cell proliferation and apoptosis resistance of low (CF1), medium (CF2) and high (CF3) floatation density fractions. EVs derived from all fractions, were internalized by renal cells, CF1 and CF2 but not CF3 fraction stimulated significant cell proliferation. CF2 also inhibited apoptosis on renal tubular cells submitted to ischemia-reperfusion injury. Comparative miRNomic and proteomic profiles reveal a cluster of miRNAs and proteins common to all three fractions and an enrichment of selected molecules related to renal regeneration in CF2 fraction. In conclusion, the CF2 fraction enriched in exosomal markers was the most active on renal tubular cell proliferation and protection from apoptosis.

Hard-to-cook bean (Phaseolus vulgaris L.) proteins hydrolyzed by alcalase and bromelain produced bioactive peptide fractions that inhibit targets of type-2 diabetes and oxidative stress.

PubMed

Oseguera-Toledo, Miguel E; Gonzalez de Mejia, Elvira; Amaya-Llano, Silvia L

2015-10-01

The objective was to evaluate the effect of bioactive peptide fractions from de-hulled hard-to-cook (HTC) bean on enzyme targets of type-2 diabetes and oxidative stress. Protein isolates from Pinto Durango and Negro 8025 beans were hydrolyzed (120min) with either alcalase® or bromelain and separated into five peptide fractions (<1, 1-3.5, 3.5-5, 5-10, and >10kDa) using an ultrafiltration membrane system. The <1kDa pinto Durango-bromelain fraction showed the best inhibition of α-amylase (49.9±1.4%), and the <1kDa pinto Durango-alcalase fraction inhibited both, α-glucosidase (76.4±0.5%), and dipeptidyl peptidase-IV (DPP-IV, 55.3±1.6%). Peptides LLSL, QQEG and NEGEAH were present in the most potent fractions. Hydrolysates and peptide fractions showed antioxidant capacity (ORAC: 159.6±2.9 to 932.6±1.1mmolTE/g) and nitric oxide inhibition (57.5±0.9 to 68.3±4.2%). Hydrolysates and fractions <1 and 1-3kDa were able to increase glucose-stimulated insulin secretion from iNS-1E cells up to 57% compared to glucose control. Hydrolysates from HTC beans inhibited enzymes related to diabetes management, being the smallest peptides (<1kDa) the most potent. HTC bean could be a source of protein to produce bioactive peptides with potential antidiabetic properties. Copyright © 2015 Elsevier Ltd. All rights reserved.