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Sample records for autoinducer-2 processing protein

  1. Processing the Interspecies Quorum-sensing Signal Autoinducer-2 (AI-2)

    Energy Technology Data Exchange (ETDEWEB)

    J Marques; P Lamosa; C Russell; R Ventura; C Maycock; M Semmelhack; S Miller; K Xavier

    2011-12-31

    The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.

  2. Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid.

    Science.gov (United States)

    Cuadra, Giancarlo A; Frantellizzi, Ashley J; Gaesser, Kimberly M; Tammariello, Steven P; Ahmed, Anika

    2016-07-01

    Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation. PMID:27350615

  3. Autoinducer 2 Affects Biofilm Formation by Bacillus cereus

    OpenAIRE

    Auger, Sandrine; Krin, Evelyne; Aymerich, Stéphane; Gohar, Michel

    2006-01-01

    Cell-free supernatants from growing Bacillus cereus strain ATCC 10987 induced luminescence in a Photorhabdus luminescens ΔluxS mutant, indicating the production of functional autoinducer 2 (AI-2). The exogenous addition of in vitro synthesized AI-2 had an inhibitory effect on biofilm formation by B. cereus and promoted release of the cells from a preformed biofilm.

  4. D-Galactose as an autoinducer 2 inhibitor to control the biofilm formation of periodontopathogens.

    Science.gov (United States)

    Ryu, Eun-Ju; Sim, Jaehyun; Sim, Jun; Lee, Julian; Choi, Bong-Kyu

    2016-09-01

    Autoinducer 2 (AI-2) is a quorum sensing molecule to which bacteria respond to regulate various phenotypes, including virulence and biofilm formation. AI-2 plays an important role in the formation of a subgingival biofilm composed mostly of Gram-negative anaerobes, by which periodontitis is initiated. The aim of this study was to evaluate D-galactose as an inhibitor of AI-2 activity and thus of the biofilm formation of periodontopathogens. In a search for an AI-2 receptor of Fusobacterium nucleatum, D-galactose binding protein (Gbp, Gene ID FN1165) showed high sequence similarity with the ribose binding protein (RbsB), a known AI-2 receptor of Aggregatibacter actinomycetemcomitans. D-Galactose was evaluated for its inhibitory effect on the AI-2 activity of Vibrio harveyi BB152 and F. nucleatum, the major coaggregation bridge organism, which connects early colonizing commensals and late pathogenic colonizers in dental biofilms. The inhibitory effect of D-galactose on the biofilm formation of periodontopathogens was assessed by crystal violet staining and confocal laser scanning microscopy in the absence or presence of AI-2 and secreted molecules of F. nucleatum. D-Galactose significantly inhibited the AI-2 activity of V. harveyi and F. nucleatum. In addition, D-galactose markedly inhibited the biofilm formation of F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia induced by the AI-2 of F. nucleatum without affecting bacterial growth. Our results demonstrate that the Gbp may function as an AI-2 receptor and that galactose may be used for prevention of the biofilm formation of periodontopathogens by targeting AI-2 activity. PMID:27572513

  5. Escherichia coli pfs Transcription: Regulation and Proposed Roles in Autoinducer-2 Synthesis and Purine Excretion▿

    OpenAIRE

    Kim, Youngbae; Lew, Chih M.; Gralla, Jay D.

    2006-01-01

    Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach sta...

  6. Autoinducer 2 Is Required for Biofilm Growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans▿

    OpenAIRE

    Shao, HanJuan; Lamont, Richard J.; Demuth, Donald R.

    2007-01-01

    Autoinducer 2 (AI-2) is required for the growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans in culture under conditions of iron limitation. However, in vivo this organism thrives in a complex multispecies biofilm that forms in the human oral cavity. In this report, we show that adherent growth of A. actinomycetemcomitans on a saliva-coated surface, but not planktonic growth under iron-replete conditions, is defective in a LuxS-deficient background. Biofilm growth of the luxS mut...

  7. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways

    Directory of Open Access Journals (Sweden)

    Wagner-Döbler Irene

    2004-09-01

    Full Text Available Abstract Background Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Recently, it has been proposed that autoinducer-2 (AI-2, a furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine (SAH to homocysteine, serves as a universal signal for interspecies communication. Results In this study, 138 completed genomes were examined for the genes involved in the synthesis and detection of AI-2. Except for some symbionts and parasites, all organisms have a pathway to recycle SAH, either using a two-step enzymatic conversion by the Pfs and LuxS enzymes or a one-step conversion using SAH-hydrolase (SahH. 51 organisms including most Gamma-, Beta-, and Epsilonproteobacteria, and Firmicutes possess the Pfs-LuxS pathway, while Archaea, Eukarya, Alphaproteobacteria, Actinobacteria and Cyanobacteria prefer the SahH pathway. In all 138 organisms, only the three Vibrio strains had strong, bidirectional matches to the periplasmic AI-2 binding protein LuxP and the central signal relay protein LuxU. The initial two-component sensor kinase protein LuxQ, and the terminal response regulator luxO are found in most Proteobacteria, as well as in some Firmicutes, often in several copies. Conclusions The genomic analysis indicates that the LuxS enzyme required for AI-2 synthesis is widespread in bacteria, while the periplasmic binding protein LuxP is only present in Vibrio strains. Thus, other organisms may either use components different from the AI-2 signal transduction system of Vibrio strains to sense the signal of AI-2, or they do not have such a quorum sensing system at all.

  8. Zoosporic plant pathogens produce bacterial autoinducer-2 that affects Vibrio harveyi quorum sensing

    Science.gov (United States)

    Kong, Ping; Lee, Bobby W.K.; Zhou, Zhaohui Sunny; Hong, Chuanxue

    2009-01-01

    The frequent co-isolation of bacteria with Phytophthora and Pythium species suggests possible interspecies communication. Zoospore free fluids (ZFF) from bacteria-free and nutrient-depleted zoospore suspensions were examined to investigate production of autoinducer-2 (AI-2), a bacterial interspecies signal molecule, by zoosporic oomycetes. ZFF from P. nicotianae, P. sojae and Py. aphanidermatum triggered luminescence of Vibrio harveyi AI-2 reporter, indicating the presence of AI-2 in zoospore extracellular products and the potential of cross-kingdom communication between oomycetes and bacteria. Production of AI-2 by zoospores was confirmed by chemical assays. These results provide new insight into the physiology and ecology of oomycetes. PMID:20002192

  9. Developing a cell-based sensor for the detection of Autoinducer-2

    Science.gov (United States)

    Servinsky, Matthew D.; Germane, Katherine; Gerlach, Elliot S.; Tsao, Chen-Yu; Byrd, Christopher M.; Sund, Christian J.; Bentley, William E.

    2013-05-01

    Bacteria use an intricate set of communication systems for sensing and interpreting environmental cues that coordinate population-based behavior. Quorum sensing is one of these systems, and it involves the production, release, and detection of small chemical signaling molecules. Recent research has revealed the role of quorum sensing molecules in the control of microbial activities such as biofilm formation. In this presentation we outline the development of a recombinant E. coli cell-based sensor for detection of the quorum sensing molecule Autoinducer-2 (AI-2), as well as engineering strategies to remove sugar and anoxic inhibition of the strain.

  10. Escherichia coli pfs transcription: regulation and proposed roles in autoinducer-2 synthesis and purine excretion.

    Science.gov (United States)

    Kim, Youngbae; Lew, Chih M; Gralla, Jay D

    2006-11-01

    Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions. PMID:16950920

  11. Autoinducer-2 of the fire blight pathogen Erwinia amylovora and other plant-associated bacteria.

    Science.gov (United States)

    Mohammadi, Mojtaba; Geider, Klaus

    2007-01-01

    Autoinducers are important for cellular communication of bacteria. The luxS gene has a central role in the synthesis of autoinducer-2 (AI-2). The gene was identified in a shotgun library of Erwinia amylovora and primers designed for PCR amplification from bacterial DNA. Supernatants of several Erwinia amylovora strains were assayed for AI-2 activity with a Vibrio harveyi mutant and were positive. Many other plant-associated bacteria also showed AI-2 activity such as Erwinia pyrifoliae and Erwinia tasmaniensis. The luxS genes of several bacteria were cloned, sequenced, and complemented Escherichia coli strain DH5alpha and a Salmonella typhimurium mutant, both defective in luxS, for synthesis of AI-2. Assays to detect AI-2 activity in culture supernatants of several Pseudomonas syringae pathovars failed, which may indicate the absence of AI-2 or synthesis of another type. Several reporter strains did not detect synthesis of an acyl homoserine lactone (AHL, AI-1) by Erwinia amylovora, but confirmed AHL-synthesis for Erwinia carotovora ssp. atroseptica and Pantoea stewartii. PMID:17092294

  12. Autoinducer-2 properties of kimchi are associated with lactic acid bacteria involved in its fermentation.

    Science.gov (United States)

    Park, Hyunjoon; Shin, Heuynkil; Lee, Kyuyeon; Holzapfel, Wilhelm

    2016-05-16

    Bacteria use the cell density-dependent quorum signalling system to regulate particular gene expressions. In food microbiology, signalling is well known for its relation to (foodborne) pathogenicity, food spoilage, and biofilm formation. Quorum quenching and inhibition are thus being considered as a feasible approach in food preservation and safety. In the case of the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2), however, it could be a different issue. Several studies have reported a luxS AI-2 synthase homologue in numerous bacteria, comprising both pathogens and beneficial strains. A recent study has shown the AI-2 signal to restore the balance of the major phyla of the gut microbiota in antibiotic-induced dysbiosis. We measured the AI-2 activity of the lactic fermented food, kimchi, and found different AI-2 signalling intensities. In order to trace the origin of the signal production, we obtained 229 lactic acid bacterial isolates from the kimchi samples, and detected the AI-2 properties of each isolate using a modified AI-2 bioluminescence assay. Our results showed isolates of dominant species of the genera Lactobacillus, Weissella and Leuconostoc which either produced or inhibited the AI-2 signal. No isolate of the dominant species Lactobacillus sakei (75 isolates) and Lactobacillus curvatus (28 isolates) showed AI-2 producing activity, while AI-2 inhibition could not be detected for any of the 31 Lactobacillus plantarum isolates. These results suggest the AI-2 activity of kimchi to result from the interaction of the associated microbial food cultures (MFCs) during fermentation. Thus far, only sparse information is available on AI-2 signalling interaction in fermented food, however, we suggest that fermented food may be a supplier of AI-2 signalling molecules via typical MFCs. PMID:26977818

  13. Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

    Directory of Open Access Journals (Sweden)

    Steven E A Christiaen

    Full Text Available In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2, and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

  14. Production of autoinducer-2 by aerobic endospore-forming bacteria isolated from the West African fermented foods.

    Science.gov (United States)

    Qian, Yang; Kando, Christine Kere; Thorsen, Line; Larsen, Nadja; Jespersen, Lene

    2015-11-01

    Autoinducer-2 (AI-2) is a quorum-sensing (QS) molecule which mediates interspecies signaling and affects various bacterial behaviors in food fermentation. Biosynthesis of AI-2 is controlled by S-ribosylhomocysteine lyase encoded by the luxS gene. The objective of this study was to investigate production of AI-2 by aerobic endospore-forming bacteria (AEB) isolated from the West African alkaline fermented seed products Mantchoua and Maari. The study included 13 AEB strains of Bacillus subtilis, B. cereus, B. altitudinis, B. amyloliquefaciens, B. licheniformis, B. aryabhattai, B. safensis, Lysinibacillus macroides and Paenibacillus polymyxa. All the tested strains harbored the luxS gene and all strains except for P. polymyxa B314 were able to produce AI-2 during incubation in laboratory medium. Production of AI-2 by AEB was growth phase dependent, showing maximum activity at the late exponential phase. AI-2 was depleted from the culture medium at the beginning of the stationary growth phase, indicating that the tested AEB possess a functional AI-2 receptor that internalizes AI-2. This study provides the evidences of QS system in Bacillus spp. and L. macroides and new knowledge of AI-2 production by AEB. This knowledge contributes to the development of QS-based strategies for better control of alkaline fermentation. PMID:26449556

  15. Production of Autoinducer 2 in Salmonella enterica Serovar Thompson Contributes to Its Fitness in Chickens but Not on Cilantro Leaf Surfaces

    OpenAIRE

    Brandl, M. T.; Miller, W. G.; Bates, A. H.; Mandrell, R E

    2005-01-01

    Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size...

  16. Potential for luxS related signalling in marine bacteria and production of autoinducer-2 in the genus Shewanella

    Directory of Open Access Journals (Sweden)

    Wagner-Döbler Irene

    2008-01-01

    Full Text Available Abstract Background The autoinducer-2 (AI-2 group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat. Results The luxS gene was not present in any of the Alphaproteobacteria (n = 71 and Bacteroidetes strains (n = 29 tested; by contrast, these bacteria harboured the sahH gene, coding for an alternative enzyme for the detoxification of S-adenosylhomocysteine (SAH in the activated methyl cycle. Within the Gammaproteobacteria (n = 76, luxS was found in all Shewanella, Vibrio and Alteromonas isolates and some Pseudoalteromonas and Halomonas species, while sahH was detected in Psychrobacter strains. A number of Gammaproteobacteria (n = 27 appeared to have neither the luxS nor the sahH gene. We then studied the production of AI-2 in the genus Shewanella using the Vibrio harveyi bioassay. All ten species of Shewanella tested produced a pronounced peak of AI-2 towards the end of the exponential growth phase in several media investigated. The maximum of AI-2 activity was different in each Shewanella species, ranging from 4% to 46% of the positive control. Conclusion The data are consistent with those of fully sequenced bacterial genomes and show that the potential for luxS related signalling is dependent on phylogenetic affiliation rather than ecological niche and is largest in certain groups of Gammaproteobacteria in the marine environment. This is the first report on AI-2

  17. Short communication: The role of autoinducer 2 (AI-2) on antibiotic resistance regulation in an Escherichia coli strain isolated from a dairy cow with mastitis.

    Science.gov (United States)

    Xue, Ting; Yu, Lumin; Shang, Fei; Li, Wenchang; Zhang, Ming; Ni, Jingtian; Chen, Xiaolin

    2016-06-01

    Extended spectrum β-lactamase (ESBL)-positive Escherichia coli is a major etiological organism responsible for bovine mastitis. The autoinducer 2 (AI-2) quorum sensing system is widely present in many species of gram-negative and gram-positive bacteria and has been proposed to be involved in interspecies communication. In E. coli model strains, the functional mechanisms of AI-2 have been well studied; however, in clinical antibiotic-resistant E. coli strains, whether AI-2 affects the expression of antibiotic resistance genes has not been reported. In this study, we report that exogenous AI-2 increased the antibiotic resistance of a clinical E. coli strain isolated from a dairy cow with mastitis by upregulating the expression of TEM-type enzyme in an LsrR (LuxS regulated repressor)-dependent manner. PMID:27060825

  18. Whey proteins utilization in food processing

    OpenAIRE

    Malá, Stanislava

    2013-01-01

    In my study entitled "Whey proteins utilization in food processing" I address functional, nutritional and physiological properties of whey and whey proteins. In the first section I describe the composition and significance of whey and I pay attention to the surfactant and physiological properties of whey proteins. The second part of my bachelor thesis deals with technological operations during whey processing, commercially accessible whey products, and finally the use of whey proteins in the ...

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

    Directory of Open Access Journals (Sweden)

    Peiqiang Yu

    2007-01-01

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

  20. Extrusion texturized dairy proteins: processing and application.

    Science.gov (United States)

    Onwulata, Charles I; Tunick, Michael H; Qi, Phoebe X

    2011-01-01

    The primary proteins in milk, casein and the whey proteins α-lactalbumin and β-lactoglobulin, have a number of health benefits and desirable functional properties. In a twin-screw extruder, mechanical shear forces, heat, and pressure cause considerable changes in the molecular structures of the dairy proteins, a process known as texturization. These changes further impart unique functional properties to dairy proteins, resulting in new protein-based food ingredients. The new functional behavior depends on the extent of texturization and the degree of structural change imparted and is controlled by adjusting parameters such as extrusion temperature and moisture level. Such texturized proteins can be used to produce puffed high-protein snacks. Softer gels and expanded structures can be made using supercritical fluid extrusion and cold extrusion, techniques that avoid elevated temperatures, minimizing possible damage to the nutritive components and functionality of the texturized dairy proteins. The uses of the texturized dairy ingredient in food products with improved functionality and enhanced nutritive profiles are presented. PMID:21504824

  1. Chemical Changes in Proteins Produced by Thermal Processing.

    Science.gov (United States)

    Dutson, T. R.; Orcutt, M. W.

    1984-01-01

    Discusses effects of thermal processing on proteins, focusing on (1) the Maillard reaction; (2) heat denaturation of proteins; (3) aggregation, precipitation, gelation, and degradation; and (4) other thermally induced protein reactions. Also discusses effects of thermal processing on muscle foods, egg proteins, fruits and vegetables, and cereal…

  2. Navigating the protein fitness landscape with Gaussian processes

    OpenAIRE

    Romero, Philip A.; Krause, Andreas; Frances H Arnold

    2013-01-01

    Knowing how protein sequence maps to function (the “fitness landscape”) is critical for understanding protein evolution as well as for engineering proteins with new and useful properties. We demonstrate that the protein fitness landscape can be inferred from experimental data, using Gaussian processes, a Bayesian learning technique. Gaussian process landscapes can model various protein sequence properties, including functional status, thermostability, enzyme activity, and ligand binding affin...

  3. Changes of protein profile during the brewing process

    OpenAIRE

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

    2012-01-01

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

  4. Measuring the Limping of Processive Motor Proteins

    Science.gov (United States)

    Zhang, Yunxin; Fisher, Michael E.

    2011-04-01

    The cells of all living creatures rely on a host of molecular scale machines to perform vital tasks. In the spirit of this special issue of J. Stat. Phys., we describe briefly the background concerning one class of these machines, namely, processive motor proteins such as, specifically, conventional kinesin, myosin V, and cytoplasmic dynein. These single-molecule motors tow cellular cargoes under load along oriented linear molecular tracks within the cell taking many hundreds of consecutive discrete steps. Experiments aimed at understanding the mechanism of the stepping process have recently led to observations of ``limping" in which alternate steps are found to be slow or fast, respectively. Reliable experimental measurements of the ``true" or intrinsic limping factor, L 0, understood as the ideal overall ratio of the longer dwell times prior to one set of steps to the shorter times for the interlaced steps, provide a route to improving appropriate biomechanochemical models. These, in turn, may help reveal and quantify details of the underlying asymmetric walking mechanisms. However, a difficulty is posed in measuring L 0 by the inescapable thermal fluctuations that act on an individual motor molecule that takes only a finite number, say, n odd and n even steps under fixed load, etc. Accordingly, we treat the stochastic issues theoretically for some basic kinetic motor models and experimental procedures, obtaining various exact bounds and explicit results for distributions and their moments. Typically for n≲10 the observed mean values, , significantly overestimate L 0. However, the medians and rescaled means, {overline{L}}n^{ *}=< Lnrangle(n-1)/n, provide better guides to the value of L 0 provided it is not too close to unity. Separately, we present figures, a table, and approximate formulas intended to assist practically those designing, undertaking, and assessing experiments on limping.

  5. Protein interactions in enzymatic processes in textiles

    OpenAIRE

    Tzanov, Tzanko; Andreaus, Juergen; Gübitz, Georg M.; Paulo, Artur Cavaco

    2003-01-01

    Enzymes are the catalysts of all reactions in living systems. These reactions are catalysed in the active sites of globular proteins. The proteins are composed by amino acids with a variety of side chains ranging from non-polar aliphatic and aromatic to acidic, basic and neutral polar. This fact allows to a globular 3D protein to create in the active site all ranges of microenvironments for catalysis. Major advances in microbial technology and genetics allow recently the broad range of ...

  6. Retinoblastoma protein: a central processing unit

    Indian Academy of Sciences (India)

    M Poznic

    2009-06-01

    The retinoblastoma protein (pRb) is one of the key cell-cycle regulating proteins and its inactivation leads to neoplastic transformation and carcinogenesis. This protein regulates critical G1-to-S phase transition through interaction with the E2F family of cell-cycle transcription factors repressing transcription of genes required for this cell-cycle check-point transition. Its activity is regulated through network sensing intracellular and extracellular signals which block or permit phosphorylation (inactivation) of the Rb protein. Mechanisms of Rb-dependent cell-cycle control have been widely studied over the past couple of decades. However, recently it was found that pRb also regulates apoptosis through the same interaction with E2F transcription factors and that Rb–E2F complexes play a role in regulating the transcription of genes involved in differentiation and development.

  7. The Online Protein Processing Resource (TOPPR): a database and analysis platform for protein processing events.

    Science.gov (United States)

    Colaert, Niklaas; Maddelein, Davy; Impens, Francis; Van Damme, Petra; Plasman, Kim; Helsens, Kenny; Hulstaert, Niels; Vandekerckhove, Joël; Gevaert, Kris; Martens, Lennart

    2013-01-01

    We here present The Online Protein Processing Resource (TOPPR; http://iomics.ugent.be/toppr/), an online database that contains thousands of published proteolytically processed sites in human and mouse proteins. These cleavage events were identified with COmbinded FRActional DIagonal Chromatography proteomics technologies, and the resulting database is provided with full data provenance. Indeed, TOPPR provides an interactive visual display of the actual fragmentation mass spectrum that led to each identification of a reported processed site, complete with fragment ion annotations and search engine scores. Apart from warehousing and disseminating these data in an intuitive manner, TOPPR also provides an online analysis platform, including methods to analyze protease specificity and substrate-centric analyses. Concretely, TOPPR supports three ways to retrieve data: (i) the retrieval of all substrates for one or more cellular stimuli or assays; (ii) a substrate search by UniProtKB/Swiss-Prot accession number, entry name or description; and (iii) a motif search that retrieves substrates matching a user-defined protease specificity profile. The analysis of the substrates is supported through the presence of a variety of annotations, including predicted secondary structure, known domains and experimentally obtained 3D structure where available. Across substrates, substrate orthologs and conserved sequence stretches can also be shown, with iceLogo visualization provided for the latter. PMID:23093603

  8. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M

    2013-01-01

    Scientific progress in the field of enzyme modification today enables the opportunity to tune a given biocatalyst for a specific industrial application. Much work has been focused on extending the substrate repertoire and altering selectivity. Nevertheless, it is clear that many new forthcoming...... opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required...

  9. Integrated process for high conversion and high yield protein PEGylation.

    Science.gov (United States)

    Pfister, David; Morbidelli, Massimo

    2016-08-01

    Over the past decades, PEGylation has become a powerful technique to increase the in vivo circulation half-life of therapeutic proteins while maintaining their activity. The development of new therapeutic proteins is likely to require further improvement of the PEGylation methods to reach even better selectivity and yield for reduced costs. The intensification of the PEGylation process was investigated through the integration of a chromatographic step in order to increase yield and conversion for the production of mono-PEGylated protein. Lysozyme was used as a model protein to demonstrate the feasibility of such approach. In the integrated reaction/separation process, chromatography was used as fractionation technique in order to isolate and recycle the unreacted protein from the PEGylated products. This allows operating the reactor with short reaction times so as to minimize the production of multi-PEGylated proteins (i.e., conjugated to more than one polymer). That is, the reaction is stopped before the desired product (i.e., the mono-PEGylated protein) can further react, thus leading to limited conversion but high yield. The recycling of the unreacted protein was then considered to drive the protein overall conversion to completion. This approach has great potential to improve processes whose yield is limited by the further reaction of the product leading to undesirable by-products. Biotechnol. Bioeng. 2016;113: 1711-1718. © 2016 Wiley Periodicals, Inc. PMID:26757029

  10. ER Protein Processing Under Oxidative Stress: Implications and Prevention.

    Science.gov (United States)

    Khalil, Mahmoud F; Valenzuela, Carlos; Sisniega, Daniella; Skouta, Rachid; Narayan, Mahesh

    2016-06-01

    Elevated levels of mitochondrial nitrosative stress have been associated with the pathogenesis of both Parkinson's and Alzheimer's diseases. The mechanism involves catalytic poisoning of the endoplasmic reticulum (ER)-resident oxidoreductase chaperone, protein disulfide isomerase (PDI), and the subsequent accumulation of ER-processed substrate proteins. Using a model system to mimic mitochondrial oxidative and nitrosative stress, we demonstrate a PDI-independent mechanism whereby reactive oxygen species (ROS) compromise regeneration rates of disulfide bond-containing ER-processed proteins. Under ROS-duress, the secretion-destined traffic adopts disulfide-exposed structures making the protein flux retrotranslocation biased. We also demonstrate that ROS-compromised protein maturation rates can be rescued by the polyphenol ellagic acid (EA). Our results are significant in that they reveal an additional mechanism which could promote neurodegenerative disorders. Furthermore, our data reveal that EA possesses therapeutic potential as a lead prophylactic agent against oxidative/nitrosative stress-related neurodegenerative diseases. PMID:26983927

  11. Evaluation of a Commercial ELISA for Detection of Ruminant Processed Animal Proteins in Non-Ruminant Processed Animal Proteins

    NARCIS (Netherlands)

    Bremer, M.G.E.G.; Margry, R.J.C.F.; Vaessen, J.C.H.; Doremalen, van A.M.H.; Palen, van der J.G.P.; Kaathoven, van R.G.C.; Kemmers-Voncken, A.E.M.; Raamsdonk, van L.W.D.

    2013-01-01

    Due to a growing aquaculture industry, demand for high-quality proteins for aquatic feeds is increasing. Non-ruminant processed animal proteins (PAPs) have shown great potential for this purpose. Safe reintroduction of non-ruminant PAPs in aqua feed requires methods that can discriminate ruminant an

  12. Autoinhibition of Mint1 adaptor protein regulates amyloid precursor protein binding and processing

    OpenAIRE

    Matos, Maria F.; Xu, Yibin; Dulubova, Irina; Otwinowski, Zbyszek; Richardson, John M.; Tomchick, Diana R.; Rizo, Josep; Ho, Angela

    2012-01-01

    Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer’s disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PT...

  13. A Novel Method for Diminishing Protein Aggregation during Denatuaration Process

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The addition of packing material for high performance hydrophobic interaction chrornatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin (α-Chy)denatured with guanidine hydrochloride (GuHCl) solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.

  14. Predicting Protein Subcellular Location Using Digital Signal Processing

    Institute of Scientific and Technical Information of China (English)

    Yu-Xi PAN; Da-Wei LI; Yun DUAN; Zhi-Zhou ZHANG; Ming-Qing XU; Guo-Yin FENG; Lin HE

    2005-01-01

    The biological functions of a protein are closely related to its attributes in a cell. With the rapid accumulation of newly found protein sequence data in databanks, it is highly desirable to develop an automated method for predicting the subcellular location of proteins. The establishment of such a predictor will expedite the functional determination of newly found proteins and the process of prioritizing genes and proteins identified by genomic efforts as potential molecular targets for drug design. The traditional algorithms for predicting these attributes were based solely on amino acid composition in which no sequence order effect was taken into account. To improve the prediction quality, it is necessary to incorporate such an effect. However, the number of possible patterns in protein sequences is extremely large, posing a formidable difficulty for realizing this goal. To deal with such difficulty, a well-developed tool in digital signal processing named digital Fourier transform (DFT) [1] was introduced. After being translated to a digital signal according to the hydrophobicity of each amino acid, a protein was analyzed by DFT within the frequency domain. A set of frequency spectrum parameters, thus obtained, were regarded as the factors to represent the sequence order effect. A significant improvement in prediction quality was observed by incorporating the frequency spectrum parameters with the conventional amino acid composition. One of the crucial merits of this approach is that many existing tools in mathematics and engineering can be easily applied in the predicting process. It is anticipated that digital signal processing may serve as a useful vehicle for many other protein science areas.

  15. Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP)☆

    OpenAIRE

    Coughlan, Kathleen; Huang, Xiangping; He, Xiangyuan; Chung, Charlotte H.Y.; Li, Guangpu; Tang, Jordan

    2013-01-01

    Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer’s disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells ex...

  16. Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

    Science.gov (United States)

    Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

    2016-01-12

    We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells. PMID:26649773

  17. Optimal separation of jojoba protein using membrane processes

    Energy Technology Data Exchange (ETDEWEB)

    Nabetani, Hiroshi; Abbott, T.P.; Kleiman, R. [National Center for Agricultural Utilization Research, Peoria, IL (United States)

    1995-05-01

    The efficiency of a pilot-scale membrane system for purifying and concentrating jojoba protein was estimated. In this system, a jojoba extract was first clarified with a microfiltration membrane. The clarified extract was diafiltrated and the protein was purified with an ultrafiltration membrane. Then the protein solution was concentrated with the ultrafiltration membrane. Permeate flux during microfiltration was essentially independent of solids concentration in the feed, in contrast with the permeate flux during ultrafiltration which was a function of protein concentration. Based on these results, a mathematical model which describes the batchwise concentration process with ultrafiltration membranes was developed. Using this model, the combination of batchwise concentration with diafiltration was optimized, and an industrial-scale process was designed. The effect of ethylenediaminetetraacetic acid (EDTA) on the performance of the membrane system was also investigated. The addition of EDTA increased the concentration of protein in the extract and improved the recovery of protein in the final products. The quality of the final product (color and solubility) was also improved. However, EDTA decreased permeate flux during ultrafiltration.

  18. Laser processing of natural mussel adhesive protein thin films

    Energy Technology Data Exchange (ETDEWEB)

    Doraiswamy, A. [Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7575 (United States); Narayan, R.J. [Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7575 (United States)]. E-mail: roger_narayan@unc.edu; Cristescu, R. [Plasma and Radiation Physics, National Institute for Lasers, Bucharest-Magurele (Romania); Mihailescu, I.N. [Plasma and Radiation Physics, National Institute for Lasers, Bucharest-Magurele (Romania); Chrisey, D.B. [United States Naval Research Laboratory, Washington, DC (United States)

    2007-04-15

    A novel laser processing technique is presented for depositing mussel adhesive protein thin films. Synthetic adhesives (e.g., acrylics, cyanoacrylates, epoxies, phenolics, polyurethanes, and silicones) have largely displaced natural adhesives in the automotive, aerospace, biomedical, electronic, and marine equipment industries over the past century. However, rising concerns over the environmental and health effects of solvents, monomers, and additives used in synthetic adhesives have led the adhesives community to seek natural alternatives. Marine mussel adhesive protein is a formaldehyde-free natural adhesive that demonstrates excellent adhesion to several classes of materials, including pure metals, metal oxides, polymers, and glasses. We have demonstrated the deposition of Mytilus edulis foot protein-1 thin films using matrix assisted pulsed laser evaporation (MAPLE). The Fourier transform infrared spectrum data suggest that the matrix assisted pulsed laser evaporation process does not cause significant damage to the chemical structure of M. edulis foot protein-1. In addition, matrix assisted pulsed laser evaporation appears to provide a better control over film thickness and film roughness than conventional solvent-based thin film processing techniques. MAPLE-deposited mussel adhesive protein thin films have numerous potential electronic, medical, and marine applications.

  19. Laser processing of natural mussel adhesive protein thin films

    International Nuclear Information System (INIS)

    A novel laser processing technique is presented for depositing mussel adhesive protein thin films. Synthetic adhesives (e.g., acrylics, cyanoacrylates, epoxies, phenolics, polyurethanes, and silicones) have largely displaced natural adhesives in the automotive, aerospace, biomedical, electronic, and marine equipment industries over the past century. However, rising concerns over the environmental and health effects of solvents, monomers, and additives used in synthetic adhesives have led the adhesives community to seek natural alternatives. Marine mussel adhesive protein is a formaldehyde-free natural adhesive that demonstrates excellent adhesion to several classes of materials, including pure metals, metal oxides, polymers, and glasses. We have demonstrated the deposition of Mytilus edulis foot protein-1 thin films using matrix assisted pulsed laser evaporation (MAPLE). The Fourier transform infrared spectrum data suggest that the matrix assisted pulsed laser evaporation process does not cause significant damage to the chemical structure of M. edulis foot protein-1. In addition, matrix assisted pulsed laser evaporation appears to provide a better control over film thickness and film roughness than conventional solvent-based thin film processing techniques. MAPLE-deposited mussel adhesive protein thin films have numerous potential electronic, medical, and marine applications

  20. Application of hydrolyzed proteins of animal origin in processed meat.

    Science.gov (United States)

    Meinert, Lene; Broge, Eva Honnens de Lichtenberg; Bejerholm, Camilla; Jensen, Kirsten

    2016-03-01

    With increasing consumer interest in functional foods, proteins from slaughterhouse side streams can offer interesting application opportunities in this respect. Worldwide, increasing numbers of people are suffering from hypertension and protein deficiency. Hydrolyzed proteins of animal origin may show ACE-inhibitory activity, which is central to the treatment of hypertension. Furthermore, the protein content of, for example, meat products increases markedly through the addition of hydrolyzed proteins, and these protein-rich products are of interest to those suffering from protein deficiency. Through a series of analyses, six selected hydrolysates were analyzed for their application potential in the Danish meat product saveloy. Hydrolyzed pig rectum and bovine diaphragm showed the highest ACE-inhibitory activities, and these activities were maintained in the processed saveloys. The ACE-inhibitory activities could not readily be explained by the amino acid profile. The content of N-compounds in the saveloys increased with increasing addition of hydrolysate, with little difference between the added hydrolysates. A sensory panel assessed the saveloys with added porcine rectum (8%), bovine diaphragm (8%), and bovine heart (4% and 8%) as having the strongest off-flavors (chemical flavor). No increase in salty taste resulting from the addition of hydrolysates was detected in the saveloys. Finally, the consumers found the saveloys too mild in flavor and recommended the addition of more spices. PMID:27004118

  1. Extraction and downstream processing of plant-derived recombinant proteins.

    Science.gov (United States)

    Buyel, J F; Twyman, R M; Fischer, R

    2015-11-01

    Plants offer the tantalizing prospect of low-cost automated manufacturing processes for biopharmaceutical proteins, but several challenges must be addressed before such goals are realized and the most significant hurdles are found during downstream processing (DSP). In contrast to the standardized microbial and mammalian cell platforms embraced by the biopharmaceutical industry, there are many different plant-based expression systems vying for attention, and those with the greatest potential to provide inexpensive biopharmaceuticals are also the ones with the most significant drawbacks in terms of DSP. This is because the most scalable plant systems are based on the expression of intracellular proteins in whole plants. The plant tissue must therefore be disrupted to extract the product, challenging the initial DSP steps with an unusually high load of both particulate and soluble contaminants. DSP platform technologies can accelerate and simplify process development, including centrifugation, filtration, flocculation, and integrated methods that combine solid-liquid separation, purification and concentration, such as aqueous two-phase separation systems. Protein tags can also facilitate these DSP steps, but they are difficult to transfer to a commercial environment and more generic, flexible and scalable strategies to separate target and host cell proteins are preferable, such as membrane technologies and heat/pH precipitation. In this context, clarified plant extracts behave similarly to the feed stream from microbes or mammalian cells and the corresponding purification methods can be applied, as long as they are adapted for plant-specific soluble contaminants such as the superabundant protein RuBisCO. Plant-derived pharmaceutical proteins cannot yet compete directly with established platforms but they are beginning to penetrate niche markets that allow the beneficial properties of plants to be exploited, such as the ability to produce 'biobetters' with tailored

  2. Subcritical Water Processing of Proteins: An Alternative to Enzymatic Digestion?

    Science.gov (United States)

    Powell, Thomas; Bowra, Steve; Cooper, Helen J

    2016-06-21

    Subcritical water is an emerging tool in the processing of bioorganic waste. Subcritical water is an environmentally benign solvent which has the potential to provide an alternative to traditional methods of protein hydrolysis without the inclusion of expensive acids or enzymes. To date, most studies on the subcritical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of subcritical water with respect to the production of peptides from three model proteins, hemoglobin, bovine serum albumin, and β-casein, and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of subcritical water (SCW) treatment on two protein post-translational modifications, disulfide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following subcritical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild subcritical water conditions (160 °C), all proteins showed favored cleavage of the Asp-X bond. The results for β-casein revealed favored cleavage of the Glu-X bond at subcritical water temperatures of 160 and 207 °C. That was similarly observed for bovine serum albumin at a subcritical water temperature of 207 °C. Subcritical water treatment results in very limited cleavage of disulfide bonds. Reduction and alkylation of proteins either prior to or post subcritical water treatment improve reported protein sequence coverages. The results for phosphoprotein β-casein show that, under mild subcritical water conditions, phosphorylation may be retained on the peptide hydrolysis products. PMID:27181872

  3. Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.

    Science.gov (United States)

    Mirambeau, Gilles; Lyonnais, Sébastien; Gorelick, Robert J

    2010-01-01

    Retroviral nucleocapsid (NC) is central to viral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NC's aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture. PMID:21045549

  4. Process considerations for protein engineering of ω-Transaminase

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson; Schwarze, Daniel; Tufvesson, Pär; Vogel, Andreas; Woodley, John

    enantio specificity (e.e). However, it is critical that this is done in parallel process development to make sure that the properties developed also fit the process requirements. As an example, ω -transaminases (EC 2.6.1.18) can be used to produce optyically pure chiral amines (with 100% theoretical yield...... how changes to a wild type transaminase through protein engineering changed the characteristics of the biocatalyst and the implications this would have on a process. A methodology for characterizing the biocatalyst was developed which was subsequently applied to the wild type and 5 mutants selected...

  5. Protein Solubility as Quality Index for Processed Soybean

    Directory of Open Access Journals (Sweden)

    Rodica Căpriţă

    2010-05-01

    Full Text Available Protein quality of soybean meal (SBM is linked to both the reduction of antinutritional factors (ANFs, and the optimization of protein digestibility. Both insufficient- and over-heating result in poor quality SBM. Inadequate heating fails to completely destroy the ANFs, which may have a detrimental impact on animal performance, while excessive heating reduces the availability of lysine via the Maillard reaction and possibly, to a lesser extent, of other amino acids. The objective of our study was to compare some biochemical laboratory procedures for assessing quality of SBM: urease index (UI, protein dispersibility index (PDI, KOH protein solubility (PS, and nitrogen solubility index (NSI. The experimental data reveal that UI is not useful to determine excessive heat treatment since additional heating has no effect on the urease index. KOH protein solubility remains high, during initial heat treatment. In marked contrast, the PDI and NSI decreased incrementally from 78% to 20% and from 97% to 60%, respectively, when heating 0 to 30 minutes. Combing the PDI test with the urease test could be useful to monitor soybean quality. SBM containing low UI (0.3 or below and high PDI (40 to 45% may indicate that the sample is definitely high quality because it has been adequately heat processed, but not overprocessed.

  6. Detection Tuna and Processed Products Based Protein and DNA Barcoding

    Directory of Open Access Journals (Sweden)

    Nuring Wulansari

    2015-11-01

    Full Text Available Tuna is the second largest fishery commodity in Indonesia after the shrimp. Since the high demand and the limited stock of tuna resulted in fraudulent chance. Authentication is required to meassure consumers regarding the accuracy of its labeling and food safety. In this study, the authentication was based on protein and DNA barcoding using cytochrome-b gene (cyt-b of the mitochondrial DNA as the target of gene. Primer of cyt b gene was designed based on the tuna species. This study aimed to identify the authenticity of tuna fresh and its processed products through protein using SDS-PAGE and DNA barcoding techniques. The phases of this research were protein electrophoresis by SDS-PAGE, DNA extraction, PCR amplification, electrophoresis and sequencing. Samples of fresh fish (Tu1, Tu2, Tu3, Tu4, and Tu5 and processed tuna (canned and steak were successfully extracted. Result showed that SDS-PAGE proved the damage of proteins in the processed tuna, so this method was not appropriate if it is used to identify the authenticity of tuna. PCR electrophoresis results showed that the samples of tuna, tuna steak, sushi, meat ball, abon, and caned tuna were successfully amplified in the range of 500-750 bp except Ka3, which was in line with the target of DNA (620 bp. Resulted sequences of Tu2, Tu3, Tu4 and Tu5 were identified according the results of morphometric namely T. albacares, while Tu1 was identified as T. obesus with homology level of 99%. Processed tunas (steak and canned tuna were identified as T. albacares, as stated on the labels.

  7. Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes

    NARCIS (Netherlands)

    Lee, Jiyeon; Retamal, Claudio; Cuitino, Loreto; Caruano-Yzermans, Amy; Shin, Jung-Eun; van Kerkhof, Peter; Marzolo, Maria-Paz; Bu, Guojun

    2008-01-01

    Accumulation of extracellular amyloid beta peptide (A beta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of A beta from APP is greatly affected by the subcellular loca

  8. Protein immobilization and detection on laser processed polystyrene surfaces

    International Nuclear Information System (INIS)

    The bovine serum albumin (BSA)-polystyrene (PS) interface layer is laser photo activated at 157 nm for site selective multiple target-protein immobilization. The 5-15 nm photon induced interface layer has different chemical, wetting, and stiffness properties than the PS photon processed surface. The irradiated areas exhibit target-protein binding, followed by localized probe-target protein detection. The photon induced chemical modification of the BSA-PS interface layer is identified by: (1) Morphological, imaging, and analysis of surface parameters with atomic force microscopy, (2) spectroscopic shift (4 cm-1), of the amide I group and formation of new C=N, NH2, C-O, C=O, and O-C=O groups following irradiation, identified with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and (3) the different hydrophilic/hydrophobic and force-distance response of the bare PS and BSA-PS surfaces. Near field edge diffraction (Fresnel) fluorescence imaging specifies the threshold photon energy and the fluence required to optically detect the protein binding on the photon induced BSA-PS interface layer. By approximating the Fresnel integrals with analytical functions, the threshold photon energy and the fluence are expressed as the sum of zero, first, and second order harmonic terms of two characteristic diffracted modes and they are specified to be 8.73x10-9 Jand623 J m-2, respectively. Furthermore, a bioarray of three probe-target proteins is fabricated with 1.5 μm spatial resolution using a 157 nm laser microstepper. The methodology eliminates the use of intermediate polymer layers between the blocking BSA protein and the PS substrate in bioarray fabrication.

  9. Protein immobilization and detection on laser processed polystyrene surfaces

    Science.gov (United States)

    Sarantopoulou, Evangelia; Petrou, Panagiota S.; Kollia, Zoe; Palles, Dimitrios; Spyropoulos-Antonakakis, Nikolaos; Kakabakos, Sotirios; Cefalas, Alkiviadis-Constantinos

    2011-09-01

    The bovine serum albumin (BSA)-polystyrene (PS) interface layer is laser photo activated at 157 nm for site selective multiple target-protein immobilization. The 5-15 nm photon induced interface layer has different chemical, wetting, and stiffness properties than the PS photon processed surface. The irradiated areas exhibit target-protein binding, followed by localized probe-target protein detection. The photon induced chemical modification of the BSA-PS interface layer is identified by: (1) Morphological, imaging, and analysis of surface parameters with atomic force microscopy, (2) spectroscopic shift (4 cm-1), of the amide I group and formation of new C=N, NH2, C-O, C=O, and O-C=O groups following irradiation, identified with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and (3) the different hydrophilic/hydrophobic and force-distance response of the bare PS and BSA-PS surfaces. Near field edge diffraction (Fresnel) fluorescence imaging specifies the threshold photon energy and the fluence required to optically detect the protein binding on the photon induced BSA-PS interface layer. By approximating the Fresnel integrals with analytical functions, the threshold photon energy and the fluence are expressed as the sum of zero, first, and second order harmonic terms of two characteristic diffracted modes and they are specified to be 8.73×10-9Jand623 J m-2, respectively. Furthermore, a bioarray of three probe-target proteins is fabricated with 1.5 μm spatial resolution using a 157 nm laser microstepper. The methodology eliminates the use of intermediate polymer layers between the blocking BSA protein and the PS substrate in bioarray fabrication.

  10. Amyloid Precursor Protein Processing in Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Adwait BHADBHADE

    2012-03-01

    Full Text Available How to Cite this Article: Bhadbhade A, Cheng DW. Amyloid Precursor Protein Processing in Alzheimer’s Disease. Iranian Journal of Child Neurology2012;6(1:1-5.Alzheimer’s disease (AD is a progressive neurodegenerative disorder and a leading cause of dementia. The AD is characterized by presence of intraneuronal tangles and extracellular plaques in the brain. The plaques are composed of dense and mostly insoluble deposits of amyloid beta peptide (Aβ, formed by sequential cleavage of the Amyloid Precursor Protein (APP, by two pathways amyloidogenic and non-amyloidogenic. Tangles are composed of paired helical fragments, which aggregate to form, microtubular protein tau. Although Aβ plaques are established to be the cause of the disease, there exist genetic factors and other pathological identifications in addition to these which are an integral part of the disease. This article gives an overview into the mechanism of APP action, genetic factors and other pathological identifications contributing to Alzheimer’s disease formation.References Brookmeyer R, Gray S, Kawas C. Projections of Alzheimer’s disease in the United States and the public health impact of delaying disease onset. American Journal of Public Health 1998;88(9:1337. Hebert LE, Scherr PA, Bienias JL, Bennett DA, Evans DA. Alzheimer disease in the US population. Arch Neurol 2003;60(8:1119-22. Möller HJ, Graeber M. The case described by Alois Alzheimer in 1911. European Archives of Psychiatry and Clinical Neuroscience 1998:248(3:111-122. Selkoe D J. (2002. Deciphering the genesis and fate of amyloid beta-protein yields novel therapies for Alzheimer disease. J Clinic Investigat 2002;110(10: 1375-82. Wolfe MS. Tau mutations in neurodegenerative diseases. J Biolog Chem 2009;284(10:6021. Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiological reviews 2001;81(2:741. Selkoe DJ. The cell biology of [beta]-amyloid precursor protein and presenilin in Alzheimer

  11. Graphics processing unit-based alignment of protein interaction networks.

    Science.gov (United States)

    Xie, Jiang; Zhou, Zhonghua; Ma, Jin; Xiang, Chaojuan; Nie, Qing; Zhang, Wu

    2015-08-01

    Network alignment is an important bridge to understanding human protein-protein interactions (PPIs) and functions through model organisms. However, the underlying subgraph isomorphism problem complicates and increases the time required to align protein interaction networks (PINs). Parallel computing technology is an effective solution to the challenge of aligning large-scale networks via sequential computing. In this study, the typical Hungarian-Greedy Algorithm (HGA) is used as an example for PIN alignment. The authors propose a HGA with 2-nearest neighbours (HGA-2N) and implement its graphics processing unit (GPU) acceleration. Numerical experiments demonstrate that HGA-2N can find alignments that are close to those found by HGA while dramatically reducing computing time. The GPU implementation of HGA-2N optimises the parallel pattern, computing mode and storage mode and it improves the computing time ratio between the CPU and GPU compared with HGA when large-scale networks are considered. By using HGA-2N in GPUs, conserved PPIs can be observed, and potential PPIs can be predicted. Among the predictions based on 25 common Gene Ontology terms, 42.8% can be found in the Human Protein Reference Database. Furthermore, a new method of reconstructing phylogenetic trees is introduced, which shows the same relationships among five herpes viruses that are obtained using other methods. PMID:26243827

  12. Features, processing states and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function

    OpenAIRE

    Mirambeau, Gilles; Lyonnais, Sébastien; Gorelick, Robert J.

    2010-01-01

    Nucleocapsid (NC) is central to retroviral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NCs aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting i...

  13. Effect of Radiation Processing on Protein Quality of Certain Legumes

    International Nuclear Information System (INIS)

    The Effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cow peas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidney beans (Phaseolus vulgaris L), and chickpeas (Cicer arietinurn L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and available lysine (AE). IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AE, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes

  14. Reverse micellar extraction for downstream processing of proteins/enzymes.

    Science.gov (United States)

    Krishna, S Hari; Srinivas, N D; Raghavarao, K S M S; Karanth, N G

    2002-01-01

    New developments in the area of downstream processing are, hopefully, to fulfill the promises of modern biotechnology. The traditional separation processes such as chromatography or electrophoresis can become prohibitively expensive unless the product is of high value. Hence, there is a need to develop efficient and cost-effective downstream processing methods. Reverse micellar extraction is one such potential and a promising liquid-liquid extraction technique, which has received immense attention for isolation and purification of proteins/enzymes in the recent times. This technique is easy to scale-up and offers continuous operation. This review, besides briefly considering important physico-chemical and biological aspects, highlights the engineering aspects including mass transfer, mathematical modeling, and technology development. It also discusses recent developments in reverse micellar extraction such as affinity based separations, enzymatic reactions in reverse micelles coupled with membrane processes, reverse micellar extraction in hollow fibers, etc. Special emphasis has been given to some recent applications of this technique. PMID:11787493

  15. Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

    Institute of Scientific and Technical Information of China (English)

    Runzhong Liu; Haibo Hou; Xuelian Yi; Shanwen Wu; Huan Zeng

    2013-01-01

    The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.

  16. Persistence of pathogenic prion protein during simulated wastewater treatment processes

    Science.gov (United States)

    Hinckley, G.T.; Johnson, C.J.; Jacobson, K.H.; Bartholomay, C.; Mcmahon, K.D.; McKenzie, D.; Aiken, Judd M.; Pedersen, J.A.

    2008-01-01

    Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP TSE) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. ?? 2008 American Chemical Society.

  17. Soybean bio-refinery platform: enzymatic process for production of soy protein concentrate, soy protein isolate and fermentable sugar syrup.

    Science.gov (United States)

    Loman, Abdullah Al; Islam, S M Mahfuzul; Li, Qian; Ju, Lu-Kwang

    2016-10-01

    Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process. PMID:27207010

  18. Biorefinery process for protein extraction from oriental mustard (Brassica juncea (L.) Czern.) using ethanol stillage

    OpenAIRE

    Ratanapariyanuch, Kornsulee; Tyler, Robert T.; Shim, Youn Young; Reaney, Martin JT

    2012-01-01

    Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs betwee...

  19. Processing and functionality of rice bran protein and peptides

    Science.gov (United States)

    Rice bran comprises approximately 10% of the rough rice kernel. Its germ is a rich source of oil; while defatted bran consists of proteins, minerals, vitamins and fibers, or the indigestible carbohydrates. About 61% of the minerals are located in the bran. The rice bran proteins have shown great pot...

  20. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel

    2014-01-01

    UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known to...... proteins involved in neuronal process extension and maintenance are both more heavily modified and more frequently regulated at a PTM level. This suggests a clear role not only for PTMs in these processes, but possibly also for heavy protein modification in general. BIOLOGICAL SIGNIFICANCE: This study...... protein-level events, this study also provides significant insight into detailed roles for individual modified proteins in the developing brain, helping to advance the understanding of the complex protein-driven processes that underlie development. Finally, the use of a novel bioinformatic analytical tool...

  1. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    Science.gov (United States)

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

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

  3. N-terminal protein processing: A comparative proteogenomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  4. DSCG binding protein and process for preparing same

    Energy Technology Data Exchange (ETDEWEB)

    Pecht, I.; Mazurek, N.

    1987-07-28

    An essentially pure protein is described consisting essentially of the protein, (CBP), present in nature in membranes of basophile cells and in mast cells, having a molecular weight of about 60,000 +- 2,000 determined by SDS polyacrylamide electrophoresis an isoelectric point of about 3.9 and an amino acid composition of about 4 units of asparagine, 3 units of threonine and serine, 3 units glycine, 2 units alanine, 2 units proline, 1 unit cysteine, 2 units valine, 1 unit methionine, 1 unit isoleucine, 2 units leucine, 1 unit tyrosine, 1 unit phenylalanine, 2 units histamine, 2 units lysine and 1 unit arginine. The protein is able to build calcium and having a calcium dependent affinity to the disodium salt of 1,2 bis(-2 carboxychromon-5-yloxy)-2-hydroxy propane (DSCG).

  5. Effects of processing on bean (Phaseolus vulgaris L.) protein quality.

    NARCIS (Netherlands)

    Poel, van der A.F.B.

    1990-01-01

    In animal production, feeding has an important impact on productivity and health of animals and feed composition is known to influence protein and energy metabolism directly. For monogastric animals complete diets are manufactured in which feed ingredients are used to supply the energy yielding and

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

    Czech Academy of Sciences Publication Activity Database

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

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

  7. Non-conventional approaches to food processing in CELSS, 1. Algal proteins: Characterization and process optimization

    Science.gov (United States)

    Nakhost, Z.; Karel, M.; Krukonis, V. J.

    1987-01-01

    Protein isolate obtained from green algae cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine make algal protein isolate a high quality component of closed ecological life support system diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical carbon dioxide resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

  8. Non-conventional approaches to food processing in CELSS. I - Algal proteins: Characterization and process optimization

    Science.gov (United States)

    Nakhost, Z.; Karel, M.; Krukonis, V. J.

    1987-01-01

    Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of CELSS diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

  9. The potential of dry fractionation processes for sustainable plant protein production

    NARCIS (Netherlands)

    Schutyser, M.A.I.; Goot, van der A.J.

    2011-01-01

    Wet fractionation processes are mainstream technology for producing plant-derived protein isolates. Unfortunately, wet fractionation involves consumption of copious amounts of water and energy. In addition, much of the (native) functionality of proteins is lost during processing. This paper reviews

  10. PROCESSING WHEY PROTEIN ISOLATE AND CORN STARCH USING A TORQUE RHEOMETER

    Science.gov (United States)

    Processing a combination of starch and protein mixtures for food or nonfood applications involves shearing and heating to produce desired texture, appearance and thermo-mechanical properties. In this study, corn starch and whey protein isolate (WPI) were processed in a torque rheometer at varying ro...

  11. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Tacchi, Jessica L; Raymond, Benjamin B A; Haynes, Paul A; Berry, Iain J; Widjaja, Michael; Bogema, Daniel R; Woolley, Lauren K; Jenkins, Cheryl; Minion, F Chris; Padula, Matthew P; Djordjevic, Steven P

    2016-02-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

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

    Directory of Open Access Journals (Sweden)

    Alisson Borges Souza

    2014-07-01

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

  13. How occasional backstepping can speed up a processive motor protein

    CERN Document Server

    Bier, M

    2008-01-01

    Fueled by the hydrolysis of ATP, the motor protein kinesin literally walks on two legs along the biopolymer microtubule. The number of accidental backsteps that kinesin takes appears to be much larger than what one would expect given the amount of free energy that ATP hydrolysis makes available. This is puzzling as more than a billion years of natural selection should have optimized the motor protein for its speed and efficiency. But more backstepping allows for the production of more entropy. Such entropy production will make free energy available. With this additional free energy, the catalytic cycle of the kinesin can be speeded up. We show how measured backstep percentages represent an optimum at which maximal net forward speed is achieved.

  14. Microtubule Associated Proteins in Plants and the Processes They Manage

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Microtubule associated proteins (MAPs) are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band,and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification,transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis, This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.

  15. Telomerase Holoenzyme Proteins and Processivity Subunit in Tetrahymena thermophila

    OpenAIRE

    Min, Bosun

    2009-01-01

    Telomeres are specialized protein-DNA structures that protect the ends of linear chromosomes, and they are maintained by the telomerase ribonucleoprotein (RNP) enzyme complex. Recombinant telomerase RNP with catalytic activity contains, at a minimum, the catalytic reverse transcriptase subunit (TERT) and the telomerase RNA (TER). However, endogenous telomerase is a much larger holoenzyme complex, with telomerase-associated subunits that contribute to RNP assembly and regulation. Telomerase-as...

  16. Detection Tuna and Processed Products Based Protein and DNA Barcoding

    OpenAIRE

    Nuring Wulansari; Mala Nurilamala; Nurjanah

    2015-01-01

    Tuna is the second largest fishery commodity in Indonesia after the shrimp. Since the high demand and the limited stock of tuna resulted in fraudulent chance. Authentication is required to meassure consumers regarding the accuracy of its labeling and food safety. In this study, the authentication was based on protein and DNA barcoding using cytochrome-b gene (cyt-b) of the mitochondrial DNA as the target of gene. Primer of cyt b gene was designed based on the tuna species. This...

  17. Polymer and protein interfacial competition in a shell production process

    Science.gov (United States)

    Willard, Emma; Randall, Greg

    2015-11-01

    We are exploring oil-in-aqueous polymer compound droplet formulations to UV polymerize into shells while in a strong AC electric field (kV/cm, 20 MHz). The electric field drives the drops to adopt a concentric configuration so that a ``perfect'' spherical shell can be polymerized with a uniform wall thickness. In our previous study of oil-in-water droplet centering, we determined that droplet stretching in the electric field was a problem, which we overcame by using protein additives to strengthen the oil/water interface. However, adding polymer to the shell fluid has been shown to weaken the droplet interface and further complicates T junction droplet generation. In this work, we study the adsorption competition between bovine serum albumin and polyethylene glycol diacrylate with the pendant drop method to generate a polymer/protein shell formulation that will resist stretching in the centering electric field. Furthermore, we explore droplet generation of polymer/protein shell formulations in a double T junction and stretching in an electric field. Work supported by General Atomics IR&D funds.

  18. Hydrophobic hydration processes. Thermal and chemical denaturation of proteins

    OpenAIRE

    Fisicaro, E.; Compari, C.; Braibanti, A.

    2011-01-01

    Abstract The hydrophobic hydration processes have been analysed under the light of a mixture model of water that is assumed to be composed by clusters (W5)I, clusters (W4)II and free water molecules WIII. The hydrophobic hydration processes can be subdivided into two Classes A and B. In the processes of Class A, the transformation A(? ?wWI? ?wWII+ ?wWIII+ cavity) takes place, with expulsion from the bulk of ?w water molecules WIII, whereas in the processes of Class B the opposite t...

  19. A cell-free expression and purification process for rapid production of protein biologics.

    Science.gov (United States)

    Sullivan, Challise J; Pendleton, Erik D; Sasmor, Henri H; Hicks, William L; Farnum, John B; Muto, Machiko; Amendt, Eric M; Schoborg, Jennifer A; Martin, Rey W; Clark, Lauren G; Anderson, Mark J; Choudhury, Alaksh; Fior, Raffaella; Lo, Yu-Hwa; Griffey, Richard H; Chappell, Stephen A; Jewett, Michael C; Mauro, Vincent P; Dresios, John

    2016-02-01

    Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value. PMID:26427345

  20. An update on the processing of high-protein rice products.

    Science.gov (United States)

    Shih, Fred F

    2003-12-01

    The component of protein in rice, at 7-9% by weight, is relatively low, but the total amount of rice protein potentially available is significant because the production of rice worldwide, at 380 million tons annually, is huge. Rice proteins are recognized as nutritional, hypoallergenic, and healthy for human consumption, and rice protein products have been in demand in recent years. However, because of difficulties in the processing, rice protein products, particularly high-protein content ones, have not been readily available. Two of the main sources of rice protein, rice bran and, to a lesser extent, broken rice kernels, have been under-used and under-priced. This report provides an update on the processing of these sources for rice proteins. Methods of protein processing are highlighted including the traditional alkaline extraction, enzyme-assisted extraction, and the novel uses of physical treatment prior to water extraction. Also discussed are effects of processing on the functional and nutritional properties of rice protein. PMID:14727771

  1. Influences of different thermal processings in milk, bovine meat and frog protein structure

    Directory of Open Access Journals (Sweden)

    Tatiana Coura Oliveira

    2013-06-01

    Full Text Available Several studies have associated the digestibility of proteins to its imunogenic potential. Though, it was objectified to evaluate the impact of the thermal processing with high and low temperatures on the proteins structure of three types of foods, by means of the digestibility in vitro and electroforesis en gel de poliacrilamida. The pasteurize was observed in such a way, firing 95 ºC during 15 minutes, how much freeze dried causes qualitative and quantitative modifications of constituent proteins of the food. The most sensible proteins to the increasing thermal processing order were beef, frog meat, and the last, cow milk.

  2. The Processing Technology of Feather Meal and Its Use as a Protein Source in Ruminant Ration

    OpenAIRE

    Wisri Puastuti

    2007-01-01

    Feather meal protein contains high level of keratin, which consists of 14% disulphide cystine, therefore feather meal protein is difficult to be digested by proteolytic enzymes. Feather meal must be processed before being used in the ration, because the digestibility of unprocessed feather meal is very low (5.8%). There are four processing methods of feather meal, i.e. physical, chemical, enzymatic and microbiological. The aim of these processings is to alleviate or break down the bonds in th...

  3. Proteins of human milk involved in immunological processes 

    OpenAIRE

    Jolanta Lis; Magdalena Orczyk-Pawiłowicz; Iwona Kątnik-Prastowska

    2013-01-01

    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum ...

  4. Processing, stability and interactions of lung surfactant protein C

    OpenAIRE

    Li, Jing

    2005-01-01

    Mature SP-C is a 4.2 kDa transmembrane protein which is uniquely expressed in the alveolar type II cell. Human SP-C is generated via multistep proteolytic cleavage of both the C-terminal and Nterminal regions of proSP-C. The function of SP-C in vivo remains unclear, but effects of SP-C on the adsorption, spreading, and stability of lipid films at an air/water interface have been documented in a number of in vitro studies. Infants with inherited deficiency of SP-B and SP-...

  5. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  6. High pressure processing of fish and protein denaturation

    OpenAIRE

    Kramer, Lene

    2013-01-01

    High pressure processing (HPP) is a relative new method for inactivating microorganisms and extend the shelf life of food. This technology has gained increasing attention as an innovative and safe way to process different types of food products. Norwegian salmon is surfing an international popularity wave of sushi, which is now found in retail stores as well as restaurants. Refrigerated storage for a few days is desirable from the producers’ point of view, while the consumers demand a texture...

  7. Hydrogen Peroxide Induced Protein Oxidation During Storage and Lyophilization Process.

    Science.gov (United States)

    Cheng, Weiqiang; Zheng, Xiaoyang; Yang, Mark

    2016-06-01

    Although the impact of hydrogen peroxide (HP) on proteins in liquid solutions has been studied extensively, the impact during lyophilization has been largely overlooked. The purpose of this work was to investigate the effect of HP on lyophilized proteins and HP removal by lyophilization. A protein formulation at 5 mg/mL and its placebo were spiked with HP up to 5.0 ppm and then lyophilized. HP concentration, protein oxidation, and aggregation were monitored before and after lyophilization, as well as during storage at 25°C. The lyophilization process removed on average 94.1% of HP from protein formulation, but only 72.5% from the placebo. There were also significant increases in protein oxidization and aggregation. The oxidation increment correlated with the decrease of HP concentration in both the protein formulation and placebo at all temperatures. Protein oxidation at different freezing temperatures was also studied in follow-up studies. Data from these studies suggest that (1) HP has a significant impact on oxidation and aggregation of protein during lyophilization; (2) significant oxidation can occur even when the protein formulation is frozen; (3) the oxidized protein is more prone to aggregation during lyophilization process. PMID:27238482

  8. Finding the "bio" in biobased products: electrophoretic identification of wheat proteins in processed products.

    Science.gov (United States)

    Robertson, George H; Hurkman, William J; Cao, Trung K; Tanaka, Charlene K; Orts, William J

    2010-04-14

    Verification of the biocontent in biobased or "green" products identifies genuine products, exposes counterfeit copies, supports or refutes content claims, and ensures consumer confidence. When the biocontent includes protein, elemental nitrogen analysis is insufficient for verification since non-protein, but nitrogen-rich, content also may be present. However, the proteins can be extracted, separated by electrophoretic methods, and detected by UV absorption, protein stain, or immunoblotting. We utilized capillary zone electrophoresis (CZE) to separate proteins in a gliadin fraction that had been dissolved in aqueous ethanol (70%) and polyacrylamide gel electrophoresis (PAGE) to separate proteins in a gliadin-plus-glutenin fraction that had been dissolved in water containing both sodium dodecyl sulfate (SDS) and a reducing agent, dithiothreitol (DTT). We sought to verify the presence of these wheat grain proteins in wheat bread, a wheat flake cereal, wheat beer, and an enclosure for an antique automobile ignition coil reputed to contain wheat gluten. Proteins extracted from commercial wheat, corn, and soy flours served as standards, and proteins from heat-altered wheat served as process condition references. This approach successfully identified wheat proteins in these products especially if the process temperature did not exceed 120 degrees C. Above this temperature attenuation was nearly complete for proteins analyzed by CZE, but wheat-like patterns could still be recognized by one- and two-dimensional PAGE. Immunoblots reacted with grain-specific antibodies confirmed the identities of the cereal component especially when the protein pattern was greatly altered by thermal modification, specific protein adsorption, or protein digestion. In addition to verifying that wheat proteins are present, the complementary use of these methods can reveal whether whole wheat gluten or merely an alcohol-soluble fraction had been used in the specific product and indicate the

  9. Protein extraction from heat-stabilized defatted rice bran. 1. Physical processing and enzyme treatments.

    Science.gov (United States)

    Tang, Shanhu; Hettiarachchy, Navam S; Shellhammer, Thomas H

    2002-12-01

    Physical processing with or without enzyme treatments on protein extraction from heat-stabilized defatted rice bran (HDRB) was evaluated. Freeze-thaw, sonication, high-speed blending, and high-pressure methods extracted 12%, 15%, 16%, and 11% protein, respectively. Sonication (0-100%, 750 W), followed by amylase and combined amylase and protease treatments, extracted 25.6-33.9% and 54.0-57.8% protein, respectively. Blending followed by amylase and protease treatment extracted 5.0% more protein than the nonblended enzymatic treatments. High-pressure treatments, 0-800 MPa, with water or amylase-protease combinations, extracted 10.5-11.1% or 61.8-66.6% protein, respectively. These results suggest that physical processing in combination with enzyme treatments can be effective in extracting protein from HDRB. PMID:12452673

  10. Proteomic Analysis Uncovers Novel Actions of the Neurosecretory Protein VGF in Nociceptive Processing

    OpenAIRE

    Riedl, Maureen S; Braun, Patrick D.; Kitto, Kelley F.; Roiko, Samuel A.; Anderson, Lorraine B.; Honda, Christopher N.; Fairbanks, Carolyn A.; Vulchanova, Lucy

    2009-01-01

    Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression following nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-...

  11. Regulation of homeostasis in the process of protein absorption from small intestine to blood

    OpenAIRE

    Akmal Yuldashev; Ravshan Rahmanov; Mukaddas Rahmatova; Margarita Tarinova; Aziza Nishanova; Gulnara Islamova

    2010-01-01

    Electron microscopic and immunоfluorescent study in rats aged 1 and 3 days after birth allowed to establish a process of absorption of protein from the small intestine into the lymph and blood. Blood homeostasis was provided by the proteins filtrated from glomerular capillaries of nephrons and reabsorbed by the epithelial cells in canaliculi of nephrons. The absorbed natural heterologous protein was depleted by lysosomes of epithelial cells of intestine and kidneys and macrophages. It support...

  12. Role 14-3-3 Protein in Regulation Some Cellular Processes

    Directory of Open Access Journals (Sweden)

    Nagam Khudhair

    2014-09-01

    Full Text Available The aim of this study to review an overview of the current information on the structure of proteins 14-3-3 and their complexes, in addition to that it provides insights into the mechanisms of their functions. The 14-3-3 proteins are from families maintain regulatory molecules expressed in all eukaryotic cells. It was discovered before thirty years, it is attributes of 14-3-3 proteins are able to connect a large number of signalling proteins are functionally diverse, including kinases, phosphatases and transmembrane receptors. 14-3-3 proteins play an important role in a variety of vital regulatory processes, such as protein regulation, apoptotic cell death and cell cycle control. In this review, we discussed the structural basis of 14-3-3 proteins, common structural features of their complexes, Phosphorylation, Cell cycle and Apoptosis.

  13. The role of the Aspergillus niger furin-type protease gene in processing of fungal proproteins and fusion proteins: Evidence for alternative processing of recombinant (fusion-) proteins

    NARCIS (Netherlands)

    Punt, P.J.; Drint-Kuijvenhoven, A.; Lokman, B.C.; Spencer, J.A.; Jeenes, D.; Archer, D.A.; Hondel, C.A.M.J.J. van den

    2003-01-01

    We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it

  14. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia

    Directory of Open Access Journals (Sweden)

    KAVEH RAHMANIFARAH

    2014-05-01

    Full Text Available Rahmanifarah K, Shabanpour B, Shaviklo AR, Aalami M. 2014. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia. Nusantara Bioscience 6: 1-6. The effect of protein extraction procedures (leached mince and heated suspension on selected properties of fish protein powder (proximate composition, pH, color, density, viscosity, fat adsorption, emulsifying capacity, emulsifying stability, foaming capacity, foaming stability, WBC, protein solubility in water, hygroscopicity, Trichloroacetic acid (TCA-soluble peptides and free sulfhydryl groups was investigated. Results showed that Fish protein powder (FPP produced by leaching mince (LM have higher protein, moisture, ash, pH, L*, viscosity, emulsion capacity, emulsion stability, foam capacity, foam stability, water binding capacity (WBC, protein solubility, hygroscopicity, TCA soluble peptides and free sulfhydryl group content than heated suspension (HS (P0.05. Overall, it was observed that high temperature during heating of suspension in HS method makes possible protein denaturation and aggregation. Consequently, based on functional, chemical and physical properties, extraction of fish protein by leaching process was found to be suitable for the production of fish protein powder.

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

    Science.gov (United States)

    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% ...

  16. Discrepancy between mRNA and protein abundance: Insight from information retrieval process in computers

    OpenAIRE

    Wang, Degeng

    2008-01-01

    Discrepancy between the abundance of cognate protein and RNA molecules is frequently observed. A theoretical understanding of this discrepancy remains elusive, and it is frequently described as surprises and/or technical difficulties in the literature. Protein and RNA represent different steps of the multi-stepped cellular genetic information flow process, in which they are dynamically produced and degraded. This paper explores a comparison with a similar process in computers - multi-step inf...

  17. Incidence of various process parameters on in vitro protein digestibility of beef meat

    OpenAIRE

    Hassoun, Ahmad; Sante-Lhoutellier, Veronique; Lebert, André; Kondjoyan, Alain; Daudin, Jean-Dominique

    2011-01-01

    Protein in vitro digestion was characterized by pepsin proteolysis of myofibrillar proteins extracted from processed beef samples using 2 descriptors of the kinetics: maximum value (ODmax) and half life time (t 1/2). An experimental fractional factorial design with 32 trials was used to investigate the effect of processes variables; it consists of 5 factors each taking 2 levels (muscle type, mincing, pH, NaCl content, cooking time) and 1 factor taking 4 levels (cooking temperature). The stati...

  18. Changes of protein profile during the brewing process

    Czech Academy of Sciences Publication Activity Database

    Benkovská, Dagmar; Flodrová, Dana; Bobálová, Janette

    Brno : Vysoké učení technické v Brně, Fakulta chemická, 2012, s. 43-48. ISBN 978-80-214-4425-6. [Studentská odborná konference Chemie a společnost 2011/2012. Brno (CZ), 16.02.2012] R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : proteomics * barley * beer * brewing process Subject RIV: CB - Analytical Chemistry, Separation http://hdl.handle.net/11104/0210046

  19. 9 CFR 95.4 - Restrictions on the importation of processed animal protein, offal, tankage, fat, glands, certain...

    Science.gov (United States)

    2010-01-01

    ... Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE EXPORTATION AND IMPORTATION... processed animal protein, offal, tankage, fat, glands, certain tallow other than tallow derivatives, and... importation of processed animal protein, offal, tankage, fat, glands, certain tallow other than...

  20. Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

    Science.gov (United States)

    Zhang, Yuanxing

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  1. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation.

    Science.gov (United States)

    Münch, Christian; Harper, J Wade

    2016-06-30

    The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans, the mitochondrial unfolded protein response (UPRmt) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis. While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression, our understanding of mammalian UPRmt is rudimentary, reflecting a lack of acute triggers for UPRmt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPRmt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8) or LON protease, which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPRmt. This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPRmt. PMID:27350246

  2. Process Optimization of Tempeh Protein Isolate from Soybean (Glycine Max Merr and Cowpea (Vigna Unguiculata Mixture

    Directory of Open Access Journals (Sweden)

    Asrul Bahar

    2015-04-01

    Full Text Available Production of ‘tempeh’ (fermented soybean protein isolates in Indonesia is still very low. It is necessary to increase the production with the alternative materials which is make by soybean and cowpea mixture. The utilization of this soybean cake (tempeh as protein isolate raw material is expecting benefits both of soybean and cowpea component which can complement each other. Protein’s content of protein isolates should be a minimum of 90% (db. Therefore, it is necessary to find the precipitation pH and the optimum level of purification in order to get tempeh protein isolate with high protein content. The analytical method used descriptive analysis. The optimal process of tempeh protein isolates from soybean and cowpea mixture conducted with pH 5 and pH 4 precipitation, the oil extraction was carried out at the beginning of the process (before extraction of protein and before the drying stage. The result showed that the tempeh protein isolate of soybean and cowpea mixing have 75.12% (db protein content. It was increasing in 20.67% to 50.16% from previous research.

  3. Processing Pisum sativum seed storage protein precursors in vitro

    Institute of Scientific and Technical Information of China (English)

    YANGLIJUN; CDOMONEY; 等

    1990-01-01

    The profile of polypeptides separated by SDS-PAGE from seed of major crop species such as pea(Pisum sativum) is complex,resulting from cleavage (processing) of precursors expressed from multiple copies of genes encoding vicilin and legumin,the major storage globulins.Translation in vitro of mRNAs hybridselected from mid-maturation pea seed RNAs by defined vicilin and legumin cDNA clones provided precursor molecules that were cleaved in vitro by a cell-free protease extract obtained from similar stage seed;the derived polypeptides were of comparable sizes to those observed in vivo.The feasibility of transcribing mRNA in vitro from a cDNA clone and cleavage in vitro of the derived translation products was established for a legumin clone,providing a method for determining polypeptide products of an expressed sequence.This approach will also be useful for characterising cleavage site requirements since modifications an readily be introduced at the DNA level.

  4. Process Control and Optimization for Heterologous Protein Production by Methylotrophic Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    GAO Minjie; SHI Zhongping

    2013-01-01

    The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression,and tremendous efforts have been made to increase heterologous protein productivity by P.pastoris in recent years.When new engineered yeast strains are constructed and are ready to use for industrial protein production,process control and optimization techniques should be applied to improve the fermentation performance in the following aspects:(1)increase recombinant cell concentrations in fermentor to high density during growth phase;(2)effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase;(3)decrease operation costs by relieving the working loads of heat-exchange and oxygen supply.This article reviews and discusses the key and commonly used techniques in heterologous protein production by P.pastoris,with the focus on optimizations of fermentation media and basic operation conditions,development of optimal glycerol feeding strategies for achieving high density cultivation of P.pastoris and effective heterologous protein induction methods by regulating specific growth rate,methanol concentration,temperatures,mixture ratio of multi-carbon substrates,etc.Metabolic analysis for recombinant protein production by P.pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.

  5. Bioenergy, protein and fibres from grass - biogas process monitoring; Bioenergie, Protein und Fasern aus Gras - Monitoring des Biogasprozesses

    Energy Technology Data Exchange (ETDEWEB)

    Baier, U.; Delavy, P.

    2003-07-01

    Starting in Summer 2001 the first full scale Swiss Bio-refinery for grass processing took up operation in Schaffhausen. Grass processing covers the production of technical fibres and protein concentrate as well as anaerobic digestion of residual slops for the production of biogas and 'green' electricity. The refinery is operated by the company Bioenergie Schaffhausen as a P+D (pilot + demonstration) project of the Swiss Federal Office of Energy. Under full load it will deliver 2,000 MWh of 'green' electricity (10% own needs) and 3,000 MWh heat (50% own needs). Prior to start up the Swiss technology holder 2B Biorefineries AG mandated the University of Applied Sciences HSW with lab scale testing of the mesophilic biogas potential and anaerobic degradability of residual grass processing slops. Nutrient limitations and possible inhibition risks were evaluated. During the initial 8 months of full scale operation of the refinery in Schaffhausen an intensive monitoring of the anaerobic digester's performance was carried out. Carbon and nitrogen mass balances have been set up and the development of the granular EGSB sludge was characterised. From operational data a set of performance values was elaborated. The first year of operation was characterised by only partial exploitation of the refinery's grass processing capacity. Furthermore the protein separation and production unit has not yet been incorporated. Consequently, the EGSB biogas reactor showed a significant hydraulic underload when compared to dimensioning basics. Raw residuals were characterised by a higher particulate protein fraction. Operational conditions for the EGSB reactor were worked out to allow stable operation at elevated load conditions and with protein separation in operation. (author)

  6. Utilization of protein-rich products from wheat-carbohydrate separation processes

    Energy Technology Data Exchange (ETDEWEB)

    Gras, P.W.; Simmonds, D.H.

    1980-09-01

    A key element in any proposal for obtaining liquid fuel from agricultural crops is the effective utilization of all by-products. Other papers in this symposium have suggested ways in which cereal straw and other crop and process wastes may be processed into useful fuel products. However, possibly the biggest challenge facing the cereal industry, if fuel from grain crops becomes a reality, is the effective use of the large amount of by-product protein which is potentially available from the process. This is even more of a challenge when the nutritional value of the combined cereal and microbial protein by-products is considered. In combination, protein from these two sources displays a desirable balance in its amino acid composition, and it should therefore be possible to upgrade it to furnish a valuable source of protein for human nutrition. Technology for spinning and texturizing vegetable proteins has been widely applied, particularly to soy proteins, while wheat gluten is already a component of many textured products on sale today. In addition, gluten derivatives having various functional properties have been developed in recent years. But what is required is the capacity to treat perhaps one million tonnes per year of combined wheat/microbial protein, and to upgrade it into an appetizing, nutritious and highly saleable food form. This could prove to be a major challenge to the industry if any serious attempt is made to obtain liquid fuels from agricultural sources such as cereal grains.

  7. Inter-laboratory validation study of two immunochemical methods for detection of processed ruminant proteins

    NARCIS (Netherlands)

    Raamsdonk, Van L.W.D.; Margry, R.J.C.F.; Kaathoven, Van R.G.C.; Bremer, M.G.E.G.

    2015-01-01

    In order to facilitate safe re-introduction of non-ruminant processed animal proteins (PAPs) in aqua feed, two immunoassays have been tested in an interlaboratory study for their capability to detect ruminant PAPs processed under European conditions. The sensitivity of the MELISA-TEK assay was im

  8. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    Energy Technology Data Exchange (ETDEWEB)

    Koskela, Harri [University of Helsinki, Finnish Institute for Verification of the Chemical Weapons Convention (VERIFIN) (Finland)], E-mail: Harri.T.Koskela@helsinki.fi; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami [University of Helsinki, Laboratory of Organic Chemistry (Finland)

    2007-04-15

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing.

  9. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    International Nuclear Information System (INIS)

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

  10. The Processing Technology of Feather Meal and Its Use as a Protein Source in Ruminant Ration

    Directory of Open Access Journals (Sweden)

    Wisri Puastuti

    2007-06-01

    Full Text Available Feather meal protein contains high level of keratin, which consists of 14% disulphide cystine, therefore feather meal protein is difficult to be digested by proteolytic enzymes. Feather meal must be processed before being used in the ration, because the digestibility of unprocessed feather meal is very low (5.8%. There are four processing methods of feather meal, i.e. physical, chemical, enzymatic and microbiological. The aim of these processings is to alleviate or break down the bonds in the keratin such as disulphide, ionic, ester and hydrogen bonds. Processed feather meal is known as hydrolyzed feather meal (HFM. Although the processing methods of feather meal produce different quality of HFM, the utilization of HFM in ruminant ration results in a better response than conventional protein resources. HFM has higher level of crude protein content (74 – 92%, rumen undegradable protein (70% and post rumiral digestibility (57 – 78% than soybean meal has. The utilization of HFM in ration up to 8% of dry matter can substitute soybean meal and the use of 2 – 3% HFM improved milk production in dairy cow and daily gain in sheep and cattle.

  11. DYNAMICS OF NITROGEN COMPOUNDS DURING MALTING PROCESS OF WHEAT WITH DIFFERENT PROTEIN LEVELS IN GRAIN

    OpenAIRE

    Марина Феликсовна Ростовская; Анастасия Николаевна Извекова; Борис Александрович Алябьев; Юрий Вадимович Приходько

    2014-01-01

    The change in the content of nitrogenous substances in the process of malting grain of the two varieties of spring wheat (Triticum aestivum L.), grown in the Primorye Territory and containing differing levels of proteins in the grain was evaluated. It is shown that higher protein wheat transferred more nitrogen materials to the roots and shoots and this results to significant loss of nitrogen compounds in the endosperm and germ. Increase of germination term decreases the weight of grain due t...

  12. DYNAMICS OF NITROGEN COMPOUNDS DURING MALTING PROCESS OF WHEAT WITH DIFFERENT PROTEIN LEVELS IN GRAIN

    Directory of Open Access Journals (Sweden)

    Марина Феликсовна Ростовская

    2014-10-01

    Full Text Available The change in the content of nitrogenous substances in the process of malting grain of the two varieties of spring wheat (Triticum aestivum L., grown in the Primorye Territory and containing differing levels of proteins in the grain was evaluated. It is shown that higher protein wheat transferred more nitrogen materials to the roots and shoots and this results to significant loss of nitrogen compounds in the endosperm and germ. Increase of germination term decreases the weight of grain due to loss of breath, which in turn increases the concentration of crude protein in the grain.

  13. Properties of the argentine anchovy and withemouth croaker muscle proteins obtained by alkali solubization process

    Directory of Open Access Journals (Sweden)

    Irene Rodrigues Freitas

    2014-07-01

    Full Text Available The aim of this study was to evaluate functional properties and microbiological characteristics of recovered proteins of anchovy (Engraulis anchoita and whitemouth croaker (Micropogonias furnieri through the process of alkaline solubilization and isoelectric precipitation, using different solubilization at pH 11 (NaOH and KOH and precipitation at pH 5.5 (HCl and H3PO4 reagents. Analyses of water holding capacity were carried out (at pH 3,5,7,9 and 11, oil holding capacity and Salmonella sp, Escherichia coli and Staphylococcus aureus. The water holding capacity was lowest at pH 5. The low value of the proteins recovered by alkaline solubilization process also indicates changes in the protein. The highest oil holding capacity was observed in whitemouth croaker concentrates (NaOH/H3PO4 at 5.6 ml oil/g protein. As for microbiological analyses, results showed no Salmonella in 25 g for all treatments and maximum count of 2.75 x 102 CFU/g for coagulase positive Staphylococcus for the muscle of anchovy concentrated. The protein concentrate obtained by combining NaOH/H3PO4 showed better functional quality. This process is an alternative to recover fish protein of low commercial value that may be used as an ingredient in foods.

  14. NifH: Structural and Mechanistic Similarities with Proteins Involved in Diverse Biological Processes

    Directory of Open Access Journals (Sweden)

    Surobhi Lahiri

    2008-01-01

    Full Text Available The NifH protein is a subunit of the nitrogenase enzyme that catalyzes the reduction of atmospheric nitrogen to ammonia. This protein contains highly conserved regions including the nucleotide binding sites, metal center ligands and the Switch I and Switch II domains. A number of proteins have structural and mechanistic similarities as well as evolutionary relationships with the NifH protein, notable among them being: light independent protochlorophyllide (Pchlide reductase (ChlL/FrxC or bChL, arsenite pump ATPase (ArsA, 2-hydroxyglutaryl dehydratase Component A (CompA involved in glutamate degradation and MinD that functions in spatial regulation of cell division. Although involved in very diverse biological processes, these proteins share an underlying common structural framework. This review mainly focuses on the structural similarities of these proteins with the NifH protein and discusses recent reports of complementation studies involving NifH and few of the proteins mentioned.

  15. Effects of power ultrasound on oxidation and structure of beef proteins during curing processing.

    Science.gov (United States)

    Kang, Da-Cheng; Zou, Yun-He; Cheng, Yu-Ping; Xing, Lu-Juan; Zhou, Guang-Hong; Zhang, Wan-Gang

    2016-11-01

    The aim of this study was to evaluate the effects of power ultrasound intensity (PUS, 2.39, 6.23, 11.32 and 20.96Wcm(-2)) and treatment time (30, 60, 90 and 120min) on the oxidation and structure of beef proteins during the brining procedure with 6% NaCl concentration. The investigation was conducted with an ultrasonic generator with the frequency of 20kHz and fresh beef at 48h after slaughter. Analysis of TBARS (Thiobarbituric acid reactive substances) contents showed that PUS treatment significantly increased the extent of lipid oxidation compared to static brining (P<0.05). As indicators of protein oxidation, the carbonyl contents were significantly affected by PUS (P<0.05). SDS-PAGE analysis showed that PUS treatment increased protein aggregation through disulfide cross-linking, indicated by the decreasing content of total sulfhydryl groups which would contribute to protein oxidation. In addition, changes in protein structure after PUS treatment are suggested by the increases in free sulfhydryl residues and protein surface hydrophobicity. Fourier transformed infrared spectroscopy (FTIR) provided further information about the changes in protein secondary structures with increases in β-sheet and decreases in α-helix contents after PUS processing. These results indicate that PUS leads to changes in structures and oxidation of beef proteins caused by mechanical effects of cavitation and the resultant generation of free radicals. PMID:27245955

  16. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham.

    Science.gov (United States)

    Gallego, Marta; Mora, Leticia; Fraser, Paul D; Aristoy, María-Concepción; Toldrá, Fidel

    2014-04-15

    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-90) providing evidence for the complexity and variability of proteolytic reactions throughout the whole process of dry-curing. Methionine oxidation has been observed in several peptides by the end of the process. The potential of some of the identified peptides to be used as biomarkers of dry-cured ham processing has also been considered. PMID:24295685

  17. Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

    Directory of Open Access Journals (Sweden)

    Henry Ryan E

    2009-03-01

    Full Text Available Abstract Background Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU. Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. Results The luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL, we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the

  18. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    Energy Technology Data Exchange (ETDEWEB)

    El-Niely, Hania F.G. [Food Irradiation Research Department, National Center for Radiation Research and Technology, Atomic Energy Authority, P.O. Box 29, Nasr City, Cairo (Egypt)]. E-mail: elniely@hotmail.com

    2007-06-15

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cowpeas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidneybeans (Phaseolus vulgaris L), and chickpeas (Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes.

  19. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    Science.gov (United States)

    El-Niely, Hania F. G.

    2007-06-01

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas ( Pisum satinum L), cowpeas ( Vigna unguiculata L.Walp), lentils ( Lens culinaris Med), kidneybeans ( Phaseolus vulgaris L), and chickpeas ( Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly ( p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes.

  20. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds

    International Nuclear Information System (INIS)

    The effects of irradiation (dose levels of 5, 7.5 and 10 kGy) on nutritive characteristics of peas (Pisum satinum L), cowpeas (Vigna unguiculata L.Walp), lentils (Lens culinaris Med), kidneybeans (Phaseolus vulgaris L), and chickpeas (Cicer arietinum L) were examined. Analyses included proximate composition, levels of anti-nutrients (phytic acid, tannins), available lysine (AL), in vitro protein digestibility (IVPD), and protein efficiency ratio (PER) in the growing rat. The results showed that moisture, crude protein, crude fat, crude fiber, and ash were unchanged by the irradiation. Radiation processing significantly (p<0.05) reduced the levels of phytic acid (PA), tannins (TN), and AL. IVPD and PER were significantly enhanced in a dose-dependent manner, relative to unirradiated control samples, for all legumes. The data sets for each legume exhibited high correlation coefficients between radiation dose and PA, TN, AL, IVPD, and PER. These results demonstrate the benefits of irradiation on the nutritional properties of these legumes

  1. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity

    International Nuclear Information System (INIS)

    This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

  2. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Turunen, S; Kaepylae, E; Kellomaeki, M [Tampere University of Technology, Department of Biomedical Engineering, PO Box 692, 33101 Tampere (Finland); Terzaki, K; Fotakis, C; Farsari, M [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), N. Plastira 100, 70013, Heraklion, Crete (Greece); Viitanen, J, E-mail: elli.kapyla@tut.fi [VTT Technical Research Centre of Finland, PO Box 1300, 33101 Tampere (Finland)

    2011-12-15

    This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

  3. Radiation processing of silk protein (Bilateral research cooperation OAEP and JAERI. December 1998 - December 2002)

    International Nuclear Information System (INIS)

    Thailand's production of silk, about 1,200 ton per year, also gives about 10% of silk waste which is expected to be recycled into new material (non-textile application) and to avoid environmental pollution. For this purpose, cooperative program 'radiation processing of silk protein' was conducted between OAEP (Thailand) and JAERI. Among the results already obtained are: radiation degradation of silk protein (fibroin) with gamma rays at 160 kGy, production of fine silk milled powder (<90 microns) by electron beam irradiation at 250-1000 kGy (dry method) using electron accelerator (1 MeV, 1 mA), use of antioxidant effect of silk protein on lipid peroxidation and antibacterial activity of irradiated silk protein powder, and wound dressing hydrogel mixed with silk protein and use of antibacterial activity of cross-linked silk protein/PVA hydrogel. Other topics of interest are gamma irradiation of anionic natural polymer solution for use as latex protein scavenger and gamma radiation degradation of chitosan for use as plant growth promoter and fungicide. (S. Ohno)

  4. Proteomic analysis of differential protein expression in early process of pancreatic regeneration in pancreatectomized rats

    Institute of Scientific and Technical Information of China (English)

    Ming YANG; Wei LIU; Chun-you WANG; Tao LIU; Feng ZHOU; Jing TAO; Yang WANG; Ming-tao LI

    2006-01-01

    Aim: A broad-range proteomic approach was applied to investigate the complexity of the mechanisms involved in pancreatic regeneration for identification of new targets of diabetes treatment and potential markers of pancreatic stem cells. Methods: A regeneration pancreatic model was induced by 90% partial pancreatectomy (Px) in rats. Changes in the protein expression in regenerating rat pancreas on the third day after Px, as compared with rats that received sham surgery, were analyzed by using 2-D gel electrophoresis (2-DE), mass spectrometry(MS), and mass fingerprinting. Results: 2-DE revealed 91 spots with at least 1.5-fold increases in expression at 3 d after pancreatectomy and 53 differentially expressed proteins that were identified by peptide mass fingerprinting (PMF). These included cell growth-related, lipid and energy metabolism-related, protein and amino acid metabolism-related proteins, and signal transduction proteins. Vimentin, CK8, L-plastin. hnRNP A2/B1, and AGAT are associated with embryogenesis and cell differentiation, and may be new potential pancreatic stem cells markers. Conclusion: The proteome profiling technique provided a broad-based and effective approach for the rapid assimilation and identification of adaptive protein changes during pancreas regeneration induced by pancreatectomy. Our data clarify the global proteome during the pancreatic proliferation and differentiation processes, which is important for better understanding of pancreatic regeneration and for discovering of protein biomarkers for pancreatic stem cells.

  5. Importance of Translational Entropy of Water in Biological Self-Assembly Processes like Protein Folding

    Directory of Open Access Journals (Sweden)

    Masahiro Kinoshita

    2009-03-01

    Full Text Available We briefly review our studies on the folding/unfolding mechanisms of proteins. In biological self-assembly processes such as protein folding, the number of accessible translational configurations of water in the system increases greatly, leading to a large gain in the water entropy. The usual view looking at only the water in the close vicinity of the protein surface is capable of elucidating neither the large entropic gain upon apoplastocyanin folding, which has recently been found in a novel experimental study, nor the pressure and cold denaturation. With the emphasis on the translational entropy of water, we are presently constructing a reliable method for predicting the native structure of a protein from its amino-acid sequence.

  6. Processing and intracellular localization of rice stripe virus Pc2 protein in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Shuling; Zhang, Gaozhan; Dai, Xuejuan; Hou, Yanling; Li, Min [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China); Liang, Jiansheng, E-mail: jsliang@yzu.edu.cn [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China); Liang, Changyong, E-mail: cyliang@yzu.edu.cn [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China)

    2012-08-01

    Rice stripe virus (RSV) belongs to the genus Tenuivirus and its genome consists of four single-stranded RNAs encoding seven proteins. Here, we have analyzed the processing and membrane association of Pc2 encoded by vcRNA2 in insect cells. The enhanced green fluorescent protein (eGFP) was fused to the Pc2 and used for the detection of Pc2 fusion proteins. The results showed that Pc2 was cleaved to produce two proteins named Pc2-N and Pc2-C. When expressed alone, either Pc2-N or Pc2-C could transport to the Endoplasmic reticulum (ER) membranes independently. Further mutagenesis studies revealed that Pc2 contained three ER-targeting domains. The results led us to propose a model for the topology of the Pc2 in which an internal signal peptide immediately followed a cleavage site, and two transmembrane regions are contained.

  7. Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

    OpenAIRE

    1984-01-01

    Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer c...

  8. Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

    Directory of Open Access Journals (Sweden)

    Yiwen Xiang

    Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

  9. Short communication: Effects of nanofiltration and evaporation on the physiochemical properties of milk protein during processing of milk protein concentrate.

    Science.gov (United States)

    Cao, Jialu; Zhang, Wei; Wu, Shaozong; Liu, Chang; Li, Yan; Li, Haimei; Zhang, Liebing

    2015-01-01

    The aim of this work was to evaluate the effects of nanofiltration and evaporation concentration technologies on the physiochemical properties of milk protein concentrate (MPC) during processing. Skim milk, ultrafiltered milk, evaporated milk, nanofiltered milk, evaporated MPC, and nanofiltered MPC samples were collected at different processing stages. Chemical composition, microstructure of casein micelles, free sulfhydryl content, and surface hydrophobicity of the samples were determined. The insolubility index of MPC was also determined. Casein micelles aggregated compactly after evaporation while surface hydrophobicity increased and free sulfhydryl content decreased in evaporated milk compared with skim milk. However, the microstructure of the casein micelles was relatively undisturbed after nanofiltration, with reduced surface hydrophobicity and free sulfhydryl content. No significant difference was found in chemical composition between the 2 MPC preparations: approximately 61.40% protein and 28.49% lactose. In addition, the particulate microstructures of both MPC were similar. However, the insolubility index of evaporated MPC was significantly (0.58mL) higher than that of nanofiltered MPC. Nanofiltration may be an effective way to improve the solubility of MPC products. PMID:25465557

  10. Antioxidant protection of proteins and lipids in processed pork loin chops through feed supplementation with avocado.

    Science.gov (United States)

    Hernández-López, Silvia H; Rodríguez-Carpena, Javier G; Lemus-Flores, Clemente; Galindo-García, Jorge; Estévez, Mario

    2016-06-01

    This study was conducted to analyze the impact of dietary avocado on the oxidative stability of lipids and proteins during pork processing. Loins from control (fed basic diet) and treated pigs (fed on avocado-supplemented diet) were roasted (102 °C/20 min) and subsequently packed in trays wrapped with oxygen-permeable films and chilled at 4 °C for 12 days. At each processing stage (raw, cooked and cooked & chilled), pork samples from both groups were analyzed for the concentration of TBARS, the loss of tryptophan and free thiols, and the formation of protein carbonyls, disulphide bonds and Schiff bases. Processing led to a depletion of tryptophan and sulfur-containing amino acids and an increase of lipid and protein oxidation products. Dietary avocado was not able to protect against the oxidation of tryptophan and thiols but cooked & chilled loins from treated pigs had significantly lower concentration of lipid and protein carbonyls than control counterparts. Likewise, dietary avocado alleviated the formation of Schiff bases during cooking. These results illustrate the benefits of dietary avocado on the oxidative stability of processed pork loins. PMID:27478235

  11. New developments in the detection and identification of processed animal proteins in feeds

    NARCIS (Netherlands)

    Raamsdonk, van L.W.D.; Holst, von C.; Baeten, V.; Berben, G.; Boix, A.; Jong, de J.

    2007-01-01

    It is generally accepted that the most likely route of infection of cattle with bovine spongiform encephalopathy (BSE) is by consumption of feeds containing low levels of processed animal proteins (PAPs). This likely route of infection resulted in feed bans, which were primarily aimed at ruminant fe

  12. PERUBAHAN ALERGENISITAS PROTEIN KACANG KEDELAI DAN KACANG BOGOR AKIBAT PENGOLAHAN DENGAN PANAS [Allergenicity Changes of Soybean and Bambara Groundnut Protein Due to Heat Processing

    Directory of Open Access Journals (Sweden)

    Nurheni Sri Palupi

    2015-12-01

    Full Text Available Legumes contain protein as a potential allergen. Heating process was expected to eliminate the protein allergen. The aim of this study was to assess the changes in molecular weight and allergenicty of soybean grobogan variety and bambara groundnut proteins due to heat processing, i.e. boiling, steaming, oven, and roasting protein isolate was prepared by pH adjusting. SDS-PAGE method was used to determine the profile of protein molecular weight and the alergenicity was determined by ELISA method. Protein molecular weight profile of grobogan soybean and bambara groundnut that have been boiled, steamed, ovened, and roasted for 30 minutes showed variations when compared to the unheated soybean and bambara groundnut protein isolate. The amount of protein detected was reduced compared with unheated soybean and bambara groundnut. The protein allergens in grobogan soybean had molecular weight 110.0, 98.3, 84.5, 67.4, and 60.2. The heat treatment for 30 minutes removed allergenicity as indicated by no detectable protein band in immunoblotting results and the smaller Optical Density value compared with unheated soybean. Thus, the allergenicity of soybean protein due to heat processing was minimized. Bambara groundnut had protein allergens with molecular weight 113.1, 59.8, and 25.2 kDa. Protein allergen with molecular weight 25.2 and 59.8 kDa were detected in bambara groundnut processed through boiling and steaming for 30 minutes, respectively, but ELISA result showed there were still protein allergen of bambara groundnut after the heat treatment for 30 minutes.

  13. Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor.

    Science.gov (United States)

    Lazar, Iulia M; Deng, Jingren; Smith, Nicole

    2016-01-01

    The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively. PMID:27078683

  14. Proteomic analysis uncovers novel actions of the neurosecretory protein VGF in nociceptive processing.

    Science.gov (United States)

    Riedl, Maureen S; Braun, Patrick D; Kitto, Kelley F; Roiko, Samuel A; Anderson, Lorraine B; Honda, Christopher N; Fairbanks, Carolyn A; Vulchanova, Lucy

    2009-10-21

    Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression after nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-inducible protein VGF (nonacronymic), a putative neuropeptide precursor. In a rodent model of nerve injury, VGF levels were increased within 24 h in both injured and uninjured DRG neurons, and the increase persisted for at least 7 d. VGF was also upregulated 24 h after hindpaw inflammation. To determine whether peptides derived from proteolytic processing of VGF participate in nociceptive signaling, we examined the spinal effects of AQEE-30 and LQEQ-19, potential proteolytic products shown previously to be bioactive. Each peptide evoked dose-dependent thermal hyperalgesia that required activation of the mitogen-activated protein kinase p38. In addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intrathecally and in the BV-2 microglial cell line when applied in vitro. In summary, our results demonstrate rapid upregulation of VGF in sensory neurons after nerve injury and inflammation and activation of microglial p38 by VGF peptides. Therefore, VGF peptides released from sensory neurons may participate in activation of spinal microglia after peripheral tissue injury. PMID:19846725

  15. Drosophila melanogaster Hsp22: a mitochondrial small heat shock protein influencing the aging process

    Directory of Open Access Journals (Sweden)

    Genevieve eMorrow

    2015-03-01

    Full Text Available Mitochondria are involved in many key cellular processes and therefore need to rely on good protein quality control (PQC. Three types of mechanisms are in place to insure mitochondrial protein integrity: reactive oxygen species (ROS scavenging by anti-oxidant enzymes, protein folding/degradation by molecular chaperones and proteases and clearance of defective mitochondria by mitophagy. Drosophila melanogaster Hsp22 is part of the molecular chaperone axis of the PQC and is characterized by its intra-mitochondrial localization and preferential expression during aging. As a stress biomarker, the level of its expression during aging has been shown to partially predict the remaining lifespan of flies. Since over-expression of this small heat shock protein (sHSP increases lifespan and resistance to stress, Hsp22 most likely has a positive effect on mitochondrial integrity. Accordingly, Hsp22 has recently been implicated in the mitochondrial unfolding protein response (mtUPR of flies. This review will summarize the key findings on D. melanogaster Hsp22 and emphasis on its links with the aging process.

  16. Malting process optimization for protein digestibility enhancement in finger millet grain.

    Science.gov (United States)

    Hejazi, Sara Najdi; Orsat, Valérie

    2016-04-01

    Finger millet (Eleusine coracana) is a nutritious, gluten-free, and drought resistant cereal containing high amounts of protein, carbohydrate, and minerals. However, bio-availability of these nutrients is restricted due to the presence of an excessive level of anti-nutrient components, mainly phytic acid, tannin, and oxalate. It has been shown that a well-designed malting/germination process can significantly reduce these anti-nutrients and consequently enhance the nutrient availability. In the present study, the effects of two important germination factors, duration and temperature, on the enhancement of in-vitro protein digestibility of finger millet were thoroughly investigated and optimized. Based on a central composite design, the grains were germinated for 24, 36, and 48 h at 22, 26, and 30 °C. For all factor combinations, protein, peptide, phytic acid, tannin, and oxalate contents were evaluated and digestibility was assessed. It was shown that during the malting/germinating process, both temperature and duration factors significantly influenced the investigated quantities. Germination of finger millet for 48 h at 30 °C increased protein digestibility from 74 % (for native grain) up to 91 %. Besides, it notably decreased phytic acid, tannin, and oxalate contents by 45 %, 46 %, and 29 %, respectively. Linear correlations between protein digestibility and these anti-nutrients were observed. PMID:27413219

  17. Cellular growth defects triggered by an overload of protein localization processes.

    Science.gov (United States)

    Kintaka, Reiko; Makanae, Koji; Moriya, Hisao

    2016-01-01

    High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes. PMID:27538565

  18. Tunable recombinant protein expression in E. coli: enabler for continuous processing?

    Science.gov (United States)

    Marschall, Lukas; Sagmeister, Patrick; Herwig, Christoph

    2016-07-01

    Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in Escherichia coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death, and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell's metabolic capacities to a maximum. Within this article, we review genetic and process technological aspects of tunable expression systems in E. coli, providing a roadmap for the industrial exploitation of the reviewed technologies. We attempt to differentiate the term "expression tuning" from its inflationary use by providing a concise definition and highlight interesting fields of application for this versatile new technology. Dependent on the type of inducer (metabolizable or non-metabolizable), different process strategies are required in order to achieve tuning. To fully profit from the benefits of tunable systems, an independent control of growth rate and expression rate is indispensable. Being able to tackle problems such as long-term culture stability and constant product quality expression tuning is a promising enabler for continuous processing in biopharmaceutical production. PMID:27170324

  19. Process technology for production and recovery of heterologous proteins with Pichia pastoris.

    Science.gov (United States)

    Jahic, Mehmedalija; Veide, Andres; Charoenrat, Theppanya; Teeri, Tuula; Enfors, Sven-Olof

    2006-01-01

    Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion. PMID:17137292

  20. The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity

    OpenAIRE

    Nandakumar, Jayakrishnan; Bell, Caitlin F.; Weidenfeld, Ina; Zaug, Arthur J.; Leinwand, Leslie A.; Cech, Thomas R.

    2012-01-01

    Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase 1–3 . Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail 4–9 . TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-bin...

  1. Denaturation processes in gamma irradiated proteins studied by differential scanning calorimetry

    International Nuclear Information System (INIS)

    Differential scanning calorimetry was applied to study the influence of gamma irradiation on the process of denaturation of proteins, occurring in water suspensions during heating. Irradiations of solid native samples and of water suspensions of the selected globular proteins were performed in air atmosphere at ambient temperature or in solid CO2, by applying 20-30 kGy, 3 kGy and 2.5 kGy doses. A decrease of peak and onset temperatures, broadening of the denaturation endothermal effects, as well as a decline in denaturation enthalpy was observed as a result of irradiation

  2. Antioxidant and functional properties of tea protein as affected by the different tea processing methods

    OpenAIRE

    Zhang, Yu; Chen, Haixia; Ning ZHANG; Ma, Lishuai

    2013-01-01

    The Box-Behnken design combined with response surface methodology was used to optimize alkali extraction of protein from tea. Three independent extraction variables (extraction time: X1; extraction temperature: X2; alkali concentration: X3) were evaluated. The antioxidant and functional properties of tea protein as affected by different tea processing were compared. The optimum conditions were: extraction time of 85 min, extraction temperature of 80 °C, and alkali concentration of 0.15 M. Und...

  3. Autoinducer-2 activity produced by bacteria found in smear of surface ripened cheeses

    DEFF Research Database (Denmark)

    Gori, Klaus; Moslehi Jenabian, Saloomeh; Purrotti, Micol;

    2011-01-01

    -2) activity using the Vibrio harveyi (BB170) bioluminescence assay. In contrast, Brevibacterium casei and Brevibacterium linens strains were not found to have AI-2 activity. When exposed to low pH and high NaCl concentrations, AI-2 activities increased between 5.0 and 11.6× for C. casei 44701, M...

  4. Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol

    2016-03-01

    Full Text Available In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA, methacryloyloxy benzophenone (MaBP, and Na-4-styrenesulfonate (SSNa was spotted onto unmodified polymethyl methacrylate (PMMA substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes.

  5. High pressure processing of meat: effects on ultrastructure and protein digestibility.

    Science.gov (United States)

    Kaur, Lovedeep; Astruc, Thierry; Vénien, Annie; Loison, Olivier; Cui, Jian; Irastorza, Marion; Boland, Mike

    2016-05-18

    The effects of high pressure processing (HPP, at 175 and 600 MPa) on the ultrastructure and in vitro protein digestion of bovine longissimus dorsi muscle meat were studied. HPP caused a significant change in the visual appearance and texture of the meat subjected to HPP at 600 MPa so that it appeared similar to cooked meat, unlike the meat subjected to HPP at 175 MPa that showed no significant visible change in the colour and texture compared to the raw meat. The muscles were subjected to digestion under simulated gastric conditions for 1 h and then under simulated small-intestinal conditions for a further 2 h. The digests were analysed using gel electrophoresis (SDS-PAGE) and ninhydrin assay for amino N. The effect of the acid conditions of the stomach alone was also investigated. Reduced SDS-PAGE results showed that pepsin-digested (60 min) HPP meats showed fewer proteins or peptides of high molecular weight than the pepsin-digested untreated meat, suggesting more breakdown of the parent proteins in HPP-treated meats. This effect was more pronounced in the muscles treated at 600 MPa. These results are in accordance with microscopy results, which showed greater changes in the myofibrillar structure after simulated gastric digestion of the sample processed at 600 MPa than at 175 MPa. Transmission electron microscopy also showed the presence of protein aggregates in the former sample, resulting probably from protein denaturation of sarcoplasmic proteins, in the subcellular space and between myofibrils; along with cell contraction (similar to that caused by heating) in the former. PMID:27143217

  6. Genomic organization of the murine G protein beta subunit genes and related processed pseudogenes.

    Science.gov (United States)

    Kitanaka, J; Wang, X B; Kitanaka, N; Hembree, C M; Uhl, G R

    2001-12-01

    The functional significance of heterotrimeric guanine nucleotide binding protein (G protein) for the many physiological processes including the molecular mechanisms of drug addiction have been described. In investigating the changes of mRNA expression after acute psychostimulant administration, we previously identified a cDNA encoding a G protein beta1 subunit (Gbeta1) that was increased up to four-fold in certain brain regions after administration of psychostimulants. The mouse Gbeta1 gene (the mouse genetic symbol, GNB1) was mapped to chromosome 4, but little was known of its genetic features. To characterize the GNB1 gene further, we have cloned and analyzed the genomic structures of the mouse GNBI gene and its homologous sequences. The GNBI gene spans at least 50 kb, and consists of 12 exons and 11 introns. The exon/intron boundaries were determined and found to follow the GT/AG rule. Exons 3-11 encode the Gbeta1 protein, and the exon 2 is an alternative, resulting in putative two splicing variants. Although intron 11 is additional for GNBI compared with GNB2 and GNB3, the intron positions within the protein coding region of GNB1, GNB2 and GNB3 are identical, suggesting that GNB1 should have diverged from the ancestral gene family earlier than the genes for GNB2 and GNB3. We also found the 5'-truncated processed pseudogenes with 71-89% similarities to GNBI mRNA sequence, suggesting that the truncated cDNA copies, which have been reverse-transcribed from a processed mRNA for GNB1, might have been integrated into several new locations in the mouse genome. PMID:11913780

  7. Disparate effects of p24alpha and p24delta on secretory protein transport and processing.

    Directory of Open Access Journals (Sweden)

    Jeroen R P M Strating

    Full Text Available BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3, -beta(1, -gamma(3 and -delta(2 is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC. METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 or p24delta(2 specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.

  8. A new process for preparation of soybean protein concentrate with hexane-aqueous ethanol mixed solvents.

    Science.gov (United States)

    Zhang, Wei-Nong; Liu, Da-Chuan

    2005-01-01

    A new process for the preparation of soybean protein concentrate (SPC) by directly extracting full-fat soy flour with a mixture of hexane and aqueous ethanol was established. Compared with conventional methods, it has some advantages, such as saving energy and reducing protein denaturation caused by heat action during solvent recovery, because this process saves one step of solvent recovery. The effects of aqueous ethanol concentration and the mixure ratio (hexane to ethanol) on the degree of protein denaturation and product quality were investigated, on the basis of which the orthogonal tests were performed. The optimum technical parameters were obtained by analyzing the results of the orthogonal tests with statistical methods. We found that SPC can be obtained by extracting full-fat soy flour under the following conditions: mixture ratio hexane: 90% ethanol, 9:1, v/v; extraction temperature, 45 degrees C; ratio of solid to solvents, (1:2 w/v); and 5 repeated extractions (15 min each time). The results of quality analysis showed that solubility of the product was improved significantly [nitrogen solubility index (NSI) 46.6%] compared with that for ethanol washing of protein concentrate (NSI 8.7%). PMID:16152943

  9. High-resolution, hybrid optical trapping methods, and their application to nucleic acid processing proteins.

    Science.gov (United States)

    Chemla, Yann R

    2016-10-01

    Optical tweezers have become a powerful tool to investigate nucleic-acid processing proteins at the single-molecule level. Recent advances in this technique have now enabled measurements resolving the smallest units of molecular motion, on the scale of a single base pair of DNA. In parallel, new instrumentation combining optical traps with other functionalities have been developed, incorporating mechanical manipulation along orthogonal directions or fluorescence imaging capabilities. Here, we review these technical advances, their capabilities, and limitations, focusing on benchmark studies of protein-nucleic acid interactions they have enabled. We highlight recent work that combines several of these advances together and its application to nucleic-acid processing enzymes. Finally, we discuss future prospects for these exciting developments. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 704-714, 2016. PMID:27225537

  10. Markovian and Non-Markovian Protein Sequence Evolution: Aggregated Markov Process Models

    OpenAIRE

    Kosiol, Carolin; Goldman, Nick

    2011-01-01

    Over the years, there have been claims that evolution proceeds according to systematically different processes over different timescales and that protein evolution behaves in a non-Markovian manner. On the other hand, Markov models are fundamental to many applications in evolutionary studies. Apparent non-Markovian or time-dependent behavior has been attributed to influence of the genetic code at short timescales and dominance of physicochemical properties of the amino acids at long timescale...

  11. Modelling Cellular Processes using Membrane Systems with Peripheral and Integral Proteins

    OpenAIRE

    Cavaliere, Matteo; Sedwards, Sean

    2006-01-01

    Membrane systems were introduced as models of computation inspired by the structure and functioning of biological cells. Recently, membrane systems have also been shown to be suitable to model cellular processes. We introduce a new model called Membrane Systems with Peripheral and Integral Proteins. The model has compartments enclosed by membranes, floating objects, objects associated to the internal and external surfaces of the membranes and also objects integral to the membranes. The floati...

  12. A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

    OpenAIRE

    Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.; Baserga, Susan J.; Hall, Traci M. Tanaka

    2014-01-01

    RNA regulation occurs at many levels including processing to mature forms, subcellular localization, and translation. RNA-binding proteins are crucial to direct and regulate these processes. Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins are RNA-binding proteins formed from eight α-helical repeats [Pumilio (PUM) repeats] that recognize specific mRNA sequences. Previous structural studies revealed characteristic curved structures and sequence specificity u...

  13. COMPLEX HYDROLYTIC PROCESSING OF PENTOSAN PLANT BIOMASS FOR PRODUCTION FURFURAL AND PROTEIN-CONTAINING FEED ADDITIVE (REVIEW

    Directory of Open Access Journals (Sweden)

    Валерий Станиславович Болтовский

    2014-10-01

    Full Text Available The review is devoted to methods of complex hydrolytic and microbial processing for the production of furfural from pentose hydrolysates and protein-containing feed additives, said process comprising bioconversion the said pentose hydrolysates and protein-containing feed additives by solid-phase fermentation of lignocellulose.

  14. Inhibition of proteolytic processing of adenoviral proteins by epsilon-aminocaproic acid and ambenum in adenovirus-infected cells.

    Science.gov (United States)

    Nosach, Lidiya; Dyachenko, Nataliya; Zhovnovataya, Valentina; Lozinskiy, Miron; Lozitsky, Victor

    2002-01-01

    Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction. PMID:12545207

  15. FKBP12 regulates the localization and processing of amyloid precursor protein in human cell lines

    Indian Academy of Sciences (India)

    Fan-Lun Liu; Ting-Yi Liu; Fan-Lu Kung

    2014-03-01

    One of the pathological hallmarks of Alzheimer’s disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (A), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer’s disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.

  16. Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution.

    Science.gov (United States)

    Urban, Michael; Vor der Brüggen, Marc; Tampé, Robert

    2016-01-01

    Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed. The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates. High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different

  17. High intakes of protein and processed meat associate with increased incidence of type 2 diabetes.

    Science.gov (United States)

    Ericson, Ulrika; Sonestedt, Emily; Gullberg, Bo; Hellstrand, Sophie; Hindy, George; Wirfält, Elisabet; Orho-Melander, Marju

    2013-03-28

    Diets high in protein have shown positive effects on short-term weight reduction and glycaemic control. However, the understanding of how dietary macronutrient composition relates to long-term risk of type 2 diabetes is limited. The aim of the present study was to examine intakes of macronutrients, fibre and protein sources in relation to incident type 2 diabetes. In total, 27 140 individuals, aged 45-74 years, from the population-based Malmö Diet and Cancer cohort, were included. Dietary data were collected with a modified diet history method, including registration of cooked meals. During 12 years of follow-up, 1709 incident type 2 diabetes cases were identified. High protein intake was associated with increased risk of type 2 diabetes (hazard ratio (HR) 1.27 for highest compared with lowest quintile; 95 % CI 1.08, 1.49; P for trend = 0.01). When protein consumption increased by 5 % of energy at the expense of carbohydrates (HR 1.20; 95 % CI 1.09, 1.33) or fat (HR 1.21; 95 % CI 1.09, 1.33), increased diabetes risk was observed. Intakes in the highest quintiles of processed meat (HR 1.16; 95 % CI 1.00, 1.36; P for trend = 0.01) and eggs (HR 1.21; 95 % CI 1.04, 1.41; P for trend = 0.02) were associated with increased risk. Intake of fibre-rich bread and cereals was inversely associated with type 2 diabetes (HR 0.84; 95 % CI 0.73, 0.98; P for trend = 0.004). In conclusion, results from the present large population-based prospective study indicate that high protein intake is associated with increased risk of type 2 diabetes. Replacing protein with carbohydrates may be favourable, especially if fibre-rich breads and cereals are chosen as carbohydrate sources. PMID:22850191

  18. Comparison of batch and continuous multi-column protein A capture processes by optimal design.

    Science.gov (United States)

    Baur, Daniel; Angarita, Monica; Müller-Späth, Thomas; Steinebach, Fabian; Morbidelli, Massimo

    2016-07-01

    Multi-column capture processes show several advantages compared to batch capture. It is however not evident how many columns one should use exactly. To investigate this issue, twin-column CaptureSMB, 3- and 4-column periodic counter-current chromatography (PCC) and single column batch capture are numerically optimized and compared in terms of process performance for capturing a monoclonal antibody using protein A chromatography. Optimization is carried out with respect to productivity and capacity utilization (amount of product loaded per cycle compared to the maximum amount possible), while keeping yield and purity constant. For a wide range of process parameters, all three multi-column processes show similar maximum capacity utilization and performed significantly better than batch. When maximizing productivity, the CaptureSMB process shows optimal performance, except at high feed titers, where batch chromatography can reach higher productivity values than the multi-column processes due to the complete decoupling of the loading and elution steps, albeit at a large cost in terms of capacity utilization. In terms of trade-off, i.e. how much the capacity utilization decreases with increasing productivity, CaptureSMB is optimal for low and high feed titers, whereas the 3-column process is optimal in an intermediate region. Using these findings, the most suitable process can be chosen for different production scenarios. PMID:26992151

  19. Heterotrimeric G-protein signaling is critical to pathogenic processes in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Dustin E Bosch

    Full Text Available Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.

  20. Accelerating large-scale protein structure alignments with graphics processing units

    Directory of Open Access Journals (Sweden)

    Pang Bin

    2012-02-01

    Full Text Available Abstract Background Large-scale protein structure alignment, an indispensable tool to structural bioinformatics, poses a tremendous challenge on computational resources. To ensure structure alignment accuracy and efficiency, efforts have been made to parallelize traditional alignment algorithms in grid environments. However, these solutions are costly and of limited accessibility. Others trade alignment quality for speedup by using high-level characteristics of structure fragments for structure comparisons. Findings We present ppsAlign, a parallel protein structure Alignment framework designed and optimized to exploit the parallelism of Graphics Processing Units (GPUs. As a general-purpose GPU platform, ppsAlign could take many concurrent methods, such as TM-align and Fr-TM-align, into the parallelized algorithm design. We evaluated ppsAlign on an NVIDIA Tesla C2050 GPU card, and compared it with existing software solutions running on an AMD dual-core CPU. We observed a 36-fold speedup over TM-align, a 65-fold speedup over Fr-TM-align, and a 40-fold speedup over MAMMOTH. Conclusions ppsAlign is a high-performance protein structure alignment tool designed to tackle the computational complexity issues from protein structural data. The solution presented in this paper allows large-scale structure comparisons to be performed using massive parallel computing power of GPU.

  1. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

    Science.gov (United States)

    Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

    2016-03-01

    Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. PMID:26944680

  2. Exit of Plasmodium sporozoites from oocysts is an active process that involves the circumsporozoite protein.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.

  3. A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins.

    Directory of Open Access Journals (Sweden)

    Eran Bosis

    Full Text Available Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

  4. PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia.

    Science.gov (United States)

    Jobling, Rebekah K; Assoum, Mirna; Gakh, Oleksandr; Blaser, Susan; Raiman, Julian A; Mignot, Cyril; Roze, Emmanuel; Dürr, Alexandra; Brice, Alexis; Lévy, Nicolas; Prasad, Chitra; Paton, Tara; Paterson, Andrew D; Roslin, Nicole M; Marshall, Christian R; Desvignes, Jean-Pierre; Roëckel-Trevisiol, Nathalie; Scherer, Stephen W; Rouleau, Guy A; Mégarbané, André; Isaya, Grazia; Delague, Valérie; Yoon, Grace

    2015-06-01

    Non-progressive cerebellar ataxias are a rare group of disorders that comprise approximately 10% of static infantile encephalopathies. We report the identification of mutations in PMPCA in 17 patients from four families affected with cerebellar ataxia, including the large Lebanese family previously described with autosomal recessive cerebellar ataxia and short stature of Norman type and localized to chromosome 9q34 (OMIM #213200). All patients present with non-progressive cerebellar ataxia, and the majority have intellectual disability of variable severity. PMPCA encodes α-MPP, the alpha subunit of mitochondrial processing peptidase, the primary enzyme responsible for the maturation of the vast majority of nuclear-encoded mitochondrial proteins, which is necessary for life at the cellular level. Analysis of lymphoblastoid cells and fibroblasts from patients homozygous for the PMPCA p.Ala377Thr mutation and carriers demonstrate that the mutation impacts both the level of the alpha subunit encoded by PMPCA and the function of mitochondrial processing peptidase. In particular, this mutation impacts the maturation process of frataxin, the protein which is depleted in Friedreich ataxia. This study represents the first time that defects in PMPCA and mitochondrial processing peptidase have been described in association with a disease phenotype in humans. PMID:25808372

  5. A new structural framework for integrating replication protein A into DNA processing machinery

    Energy Technology Data Exchange (ETDEWEB)

    Brosey, Chris; Yan, Chunli; Tsutakawa, Susan; Heller, William; Rambo, Robert; Tainer, John; Ivanov, Ivaylo; Chazin, Walter

    2013-01-17

    By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

  6. Study on Processing Technology and a Complex Stabilizer for Peanut Protein Beverage

    Directory of Open Access Journals (Sweden)

    Xihong Zhao

    2015-08-01

    Full Text Available The development of the processing technology and a complex stabilizer for the peanut protein beverage processed was presented in this study. The suitable peeling conditions for peanut were: to made it soak with soft water containing 5% NaHCO3 for 12 h. The best homogenizing temperature, pressure and times were 75C, 30MPa and twice. The sterilization condition of 121C and 15 min was the foundation to achieve the best stability. The composition of the stabilizer was optimized by uniform design combined with regression analysis based on sensory evaluation, which was achieved using fuzzy comprehensive evaluation method. The 100 mL of peanut protein beverage added with 0.4 g of sodium carboxymethyl cellulose (CMC-Na, 0.2 g of sodium alginate and 0.6 g of gelatin displayed good stability. CMC-Na amount had the largest effect on beverage stability and the effect of gelatin amount was the smallest. The peanut protein beverage with added optimized complex stabilizer was medium preference grade.

  7. Amyloid-beta Alzheimer targets — protein processing, lipid rafts, and amyloid-beta pores

    Science.gov (United States)

    Arbor, Sage C.; LaFontaine, Mike; Cumbay, Medhane

    2016-01-01

    Amyloid beta (Aβ), the hallmark of Alzheimer’s Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review.

  8. Characterization of structural and functional properties of fish protein hydrolysates from surimi processing by-products.

    Science.gov (United States)

    Liu, Yongle; Li, Xianghong; Chen, Zhijun; Yu, Jian; Wang, Faxiang; Wang, Jianhui

    2014-05-15

    Structural and functional properties of fish protein hydrolysates with different degrees of hydrolysis (DH) from surimi processing by-products, prepared by Protamex and Alcalase, were evaluated. As the DH increased, the zeta potentials of the hydrolysates increased (p>0.05). The surface hydrophobicity of the hydrolysates was significantly affected by DH (phydrolysate with DH 10%, prepared by Protamex, contained more large protein molecules than did the others. Hydrolysis by both enzymes increased solubility to more than 65% over a wide pH range (pH 2-10). The interfacial activities of hydrolysates decreased with increasing DH (phydrolysate with DH 10%, prepared by Protamex, exhibited the best interfacial properties among all of the samples. Thermal properties were also affected by the hydrolysis. The results reveal that structures and functionalities of the hydrolysates were determined both by DH and enzyme type employed. PMID:24423557

  9. Amyloid-beta Alzheimer targets - protein processing, lipid rafts, and amyloid-beta pores.

    Science.gov (United States)

    Arbor, Sage C; LaFontaine, Mike; Cumbay, Medhane

    2016-03-01

    Amyloid beta (Aβ), the hallmark of Alzheimer's Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aβ fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aβ aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aβ aggregates, and current therapeutics are summarized in this review. PMID:27505013

  10. Insights into biological information processing: structural and dynamical analysis of a human protein signalling network

    International Nuclear Information System (INIS)

    We present an investigation on the structural and dynamical properties of a 'human protein signalling network' (HPSN). This biological network is composed of nodes that correspond to proteins and directed edges that represent signal flows. In order to gain insight into the organization of cell information processing this network is analysed taking into account explicitly the edge directions. We explore the topological properties of the HPSN at the global and the local scale, further applying the generating function formalism to provide a suitable comparative model. The relationship between the node degrees and the distribution of signals through the network is characterized using degree correlation profiles. Finally, we analyse the dynamical properties of small sub-graphs showing high correlation between their occurrence and dynamic stability

  11. Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts

    OpenAIRE

    Zhelyazkova, P.; Hammani, K.; M. Rojas; Voelker, R.; Vargas-Suarez, M.; Boerner, T.; Barkan, A

    2011-01-01

    Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5'- and 3'-exonucleases, resulting in processed RNAs with PPR10 bound at the 5'- or 3'-end. In this study, we prov...

  12. Modified fabrication process of protein chips using a short-chain self-assembled monolayer.

    Science.gov (United States)

    Jang, Ling-Sheng; Keng, Hao-Kai

    2008-04-01

    In previous work a short chain SAM, 4,4-Dithiodibutyric Acid (DTBA) was found to be a thin monolayer in protein chips. However, obtaining uniform fluorescent intensity remains difficult because water-soluble carbodiimides (EDC) in an aqueous system cause the hydrolysis of N-hydroxysuccinimide ester (NHS esters). The hydrolysis of NHS esters reduces coupling yields and therefore reduces the fluorescent intensity of protein chips. The NHS can increase the stability of active intermediate resulting from the reaction of EDC and NHS, but the ratio of the concentration of EDC to that of NHS strongly affects this stability. The effects of the solvents used in the washing step are studied to solve this problem. The results reveal that PBST (PBS + 5% Tween20) is more effective in reducing the hydrolysis of NHS esters than deionized water. Additionally, the effects of 3:1 and 5:2 EDC/NHS ratios on the chips are examined. The 3:1 EDC/NHS ratio yields a higher fluorescent intensity than the 5:2 ratio. The effects on the chips of dissolving EDC in DI water, DI water + 0.1 M MES and alcohol are also investigated. The results show that alcohol provides higher fluorescent intensity than other solvents and the reaction time of 4 h yields a high fluorescent intensity with 3:1 EDC/NHS ratio. A modified fabrication process of protein chips using 4,4-DTBA is developed. In this work, 160 mM 4,4-DTBA is used as a self-assembled monolayer in the fabrication of protein chips. Experiments to characterize 4,4-DTBA are performed by contact angle goniometry and Fourier transform infrared spectroscopy (FTIR). Furthermore, the immobilized protein A-FITC (fluorescein isothiocyanate) is adopted in fluorescent assays. PMID:17849186

  13. Detection of Prion Proteins and TSE Infectivity in the Rendering and Biodiesel Manufacture Processes

    Energy Technology Data Exchange (ETDEWEB)

    Brown, R.; Keller, B.; Oleschuk, R. [Queen' s University, Kingston, Ontario (Canada)

    2007-03-15

    This paper addresses emerging issues related to monitoring prion proteins and TSE infectivity in the products and waste streams of rendering and biodiesel manufacture processes. Monitoring is critical to addressing the knowledge gaps identified in 'Biodiesel from Specified Risk Material Tallow: An Appraisal of TSE Risks and their Reduction' (IEA's AMF Annex XXX, 2006) that prevent comprehensive risk assessment of TSE infectivity in products and waste. The most important challenge for monitoring TSE risk is the wide variety of sample types, which are generated at different points in the rendering/biodiesel production continuum. Conventional transmissible spongiform encephalopathy (TSE) assays were developed for specified risk material (SRM) and other biological tissues. These, however, are insufficient to address the diverse sample matrices produced in rendering and biodiesel manufacture. This paper examines the sample types expected in rendering and biodiesel manufacture and the implications of applying TSE assay methods to them. The authors then discuss a sample preparation filtration, which has not yet been applied to these sample types, but which has the potential to provide or significantly improve TSE monitoring. The main improvement will come from transfer of the prion proteins from the sample matrix to a matrix compatible with conventional and emerging bioassays. A second improvement will come from preconcentrating the prion proteins, which means transferring proteins from a larger sample volume into a smaller volume for analysis to provide greater detection sensitivity. This filtration method may also be useful for monitoring other samples, including wash waters and other waste streams, which may contain SRM, including those from abattoirs and on-farm operations. Finally, there is a discussion of emerging mass spectrometric methods, which Prusiner and others have shown to be suitable for detection and characterisation of prion proteins (Stahl

  14. Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.

    Science.gov (United States)

    de Amorim Araújo, Juliana; Ferreira, Túlio César; Rubini, Marciano Régis; Duran, Ana Gilhema Gomez; De Marco, Janice Lisboa; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2015-12-01

    The term cellulase refers to any component of the enzymatic complex produced by some fungi, bacteria and protozoans which act serially or synergistically to catalyze the cleavage of cellulosic materials. Cellulases have been widely used in many industrial applications ranging from food industry to the production of second generation ethanol. In an effort to develop new strategies to minimize the costs of enzyme production we describe the development of a Pichia pastoris strain able to coproduce two different cellulases. For that purpose the eglII (endoglucanase II) and cbhII (cellobiohydrolase II) genes from Trichoderma reesei were fused in-frame separated by the self-processing 2A peptide sequence from the foot-and-mouth disease virus. The protein fusion construct was placed under the control of the strong inducible AOX1 promoter. Analysis of culture supernatants from methanol-induced yeast transformants showed that the protein fusion was effectively processed. Enzymatic assay showed that the processed enzymes were fully functional with the same catalytic properties of the individual enzymes produced separately. Furthermore, when combined both enzymes acted synergistically on filter paper to produce cellobiose as the main end-product. Based on these results we propose that P. pastoris should be considered as an alternative platform for the production of cellulases at competitive costs. PMID:26698316

  15. Process, cost modeling and simulations for integrated project development of biomass for fuel and protein

    International Nuclear Information System (INIS)

    The construction of the models for biomass project development are described. These models, first constructed using QPRO electronic spread sheet for Windows, are now being developed with the aid of visual and object oriented program as tools using DELPHI V.1 for windows and process simulator SUPERPRO, V.2.7 Intelligent Inc. These models render the process development problems with economic objectives to be solved very rapidly. The preliminary analysis of cost and investments of biomass utilisation projects which are included for this study are: steam, ammonia, carbon dioxide and alkali pretreatment process, methane gas production using anaerobic digestion process, aerobic composting, ethanol fermentation and distillation, effluent treatments using high rate algae production as well as cogeneration of energy for drying. The main project under developments are the biomass valuation projects with the elephant (Napier) grass, sugar cane bagasse and microalgae, using models for mass balance, equipment and production cost. The sensibility analyses are carried out to account for stochastic variation of the process yield, production volume, price variations, using Monte Carlo method. These models allow the identification of economical and scale up problems of the technology. The results obtained with few preliminary project development with few case studies are reported for integrated project development for fuel and protein using process and cost simulation models. (author)

  16. Structural determinants of stability to proteolysis, processing and impact on allergenic potential of non-specific lipid transfer proteins

    OpenAIRE

    Abdullah, Syed Umer

    2012-01-01

    Lipid transfer proteins (LTPs) are a class of low molecular weight hydrophobic conserved proteins comprising four intramolecular disulphide bonds making the structure very resistant to proteolysis and harsh food processing conditions. These proteins are identified as strong allergens sensitizing through the gut and share epitopes with LTPs from closely related species. Peach LTP, Pru p 3 is the primary sensitizer in the Mediterranean area being the most frequent food allergen. Wheat LTP, Tri ...

  17. TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements

    OpenAIRE

    Franks, Tobias M.; Lykke-Andersen, Jens

    2007-01-01

    In mammalian cells, mRNAs with AU-rich elements (AREs) are targeted for translational silencing and rapid degradation. Here we present evidence that in human cells the proteins Tristetraprolin (TTP) and BRF-1 deliver ARE-mRNAs to processing bodies (PBs), cytoplasmic assemblies of mRNAs, and associated factors that promote translational silencing and mRNA decay. First, depletion of endogenous TTP and BRF proteins, or overexpression of dominant-negative mutant TTP proteins, impairs the localiza...

  18. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    Directory of Open Access Journals (Sweden)

    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  19. Entropic formulation for the protein folding process: hydrophobic stability correlates with folding rates

    CERN Document Server

    Molin, J P Dal

    2016-01-01

    We assume that the protein folding process follows two autonomous steps: the conformational search for the native, mainly ruled by the hydrophobic effect; and, the final adjustment stage, which eventually gives stability to the native. Our main tool of investigation is a 3D lattice model provided with a ten-letter alphabet, the stereochemical model. This model was conceived for Monte Carlo (MC) simulations when one keeps in mind the kinetic behavior of protein-like chains in solution. In order to characterize the folding characteristic time ({\\tau}) by two distinct sampling methods, first we present two sets of 10^{3} MC simulations for a fast protein-like sequence. For these sets of folding times, {\\tau} and {\\tau}_{q} were obtained with the application of the standard Metropolis algorithm (MA), and a modified algorithm (M_{q}A). The results for {\\tau}_{q}reveal two things: i) the hydrophobic chain-solvent interactions plus a set of inter-residues steric constraints are enough to emulate the first stage of t...

  20. Oxidative processes during enzymatic hydrolysis of cod protein and their influence on antioxidant and immunomodulating ability.

    Science.gov (United States)

    Halldorsdottir, Sigrun M; Sveinsdottir, Holmfridur; Freysdottir, Jona; Kristinsson, Hordur G

    2014-01-01

    Fish protein hydrolysates (FPH) have many desirable properties, however heating and shifts in pH can cause oxidation during enzymatic hydrolysis. The objective was to investigate oxidative processes during enzymatic hydrolysis of fish protein and the impact of oxidation on the antioxidant and immunomodulating ability of FPH. Protease P "Amano" 6 was used to hydrolyze cod protein in the presence and absence of pro-oxidants at pH 8 and 36°C to achieve 20% degree of hydrolysis. Results from thiobarbituric acid reactive substances (TBARS) and sensory analysis indicate that oxidation can develop rapidly during hydrolysis. A cellular antioxidant assay using a HepG2 cell model indicated a negative impact of oxidation products on antioxidant properties of the FPH while results obtained in chemical assays showed a negligible impact. Results from a dendritic cell model indicating that oxidation products may affect anti-inflammatory activity in the body. This study provides important information regarding bioactive FPH. PMID:24001832

  1. A Directed Binding Mechanism of Processive Motion for the Kinesin Motor Protein Families

    International Nuclear Information System (INIS)

    A novel physical mechanism is discussed for the processive propagation of two-headed motor proteins such as kinesin along protein filaments. Our model uses the fact that the binding of each head must be directionality oriented to the protein filament. The binding sites are realized by a 2D periodic potential due to the filament's surface. The deviation of the geometry of the kinesin from the relaxed state to the state where both motor domains are simultaneously bound to the filament results in an internal stress of the molecule. Un-binding of one of the motor domains from the filament, which is due to the release of chemical energy from ATP hydrolysis, results in a mechanical movement until the relaxed state is reached again. We develop a simple mathematical and mechanical model in which directed binding of the heads to the filament results in a directed twist away from its relaxed state of the molecule, occurring probably in the neck linker region. Un-binding of the head from the filament relaxes the twist and defines the propagation direction. We show that there must be at least one torsional spring for every head to store elastic energy. It is the internal structure both of the relaxed and tensed-up state that defines the walking direction of kinesin. Calculations based on the model are in good quantitative agreement with experimental observations. (author)

  2. A Directed Binding Mechanism of Processive Motion for the Kinesin Motor Protein Families

    Science.gov (United States)

    Bolterauer, H.; Tuszynski, J. A.; Unger, E.

    2006-05-01

    A novel physical mechanism is discussed for the processive propagation of two-headed motor proteins such as kinesin along protein filaments. Our model uses the fact that the binding of each head must be directionality oriented to the protein filament. The binding sites are realized by a 2D periodic potential due to the filament's surface. The deviation of the geometry of the kinesin from the relaxed state to the state where both motor domains are simultaneously bound to the filament results in an internal stress of the molecule. Un-binding of one of the motor domains from the filament, which is due to the release of chemical energy from ATP hydrolysis, results in a mechanical movement until the relaxed state is reached again. We develop a simple mathematical and mechanical model in which directed binding of the heads to the filament results in a directed twist away from its relaxed state of the molecule, occurring probably in the neck linker region. Un-binding of the head from the filament relaxes the twist and defines the propagation direction. We show that there must be at least one torsional spring for every head to store elastic energy. It is the internal structure both of the relaxed and tensed-up state that defines the walking direction of kinesin. Calculations based on the model are in good quantitative agreement with experimental observations.

  3. AcEST: DK958373 [AcEST

    Lifescience Database Archive (English)

    Full Text Available d protein OS=Physcomitrella patens subsp. patens Align length 204 Score (bit) 115.0 E-value 2.0e-24 Repor...EVEACAKRLH 277 >sp|Q2PBM2|LSRC_PHOTE Autoinducer 2 import system permease protein lsrC OS=Photorhabdus tem...9A6|U496D_ORYSI UPF0496 protein 4 OS=Oryza sativa subsp. i... 45 3e-04 sp|Q2PBM2|LSRC_PHOTE Autoinducer 2 import system... permease protei... 40 0.009 sp|Q2PBL9|LSRC_PHOLU Autoinducer 2 import system permease protei... ...39 0.027 sp|Q7N2D8|LSRC_PHOLL Autoinducer 2 import system permease protei... 39 0.027 sp|A4TQL6|LSRC_YERPP Autoinducer 2 impor

  4. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

    Science.gov (United States)

    Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

    2016-01-01

    Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes. PMID:27383650

  5. Effects of Industrial Heating Processes of Milk-Based Enteral Formulas on Site-Specific Protein Modifications and Their Relationship to in Vitro and in Vivo Protein Digestibility.

    Science.gov (United States)

    Wada, Yasuaki; Lönnerdal, Bo

    2015-08-01

    Heat treatments are applied to milk and dairy products to ensure their microbiological safety and shelf lives. Types of heating processes may have different effects on protein modifications, leading to different protein digestibility. In this study, milk-based liquid nutritional formulas (simulating enteral formulas) were subjected to steam injection ultra-high-temperature treatment or in-can sterilization, and the formulas were investigated by proteomic methods and in vitro and in vivo digestion assays. Proteomic analyses revealed that in-can sterilization resulted in higher signals for N(ε)-carboxymethyllysine and dephosphorylation of Ser residues in major milk proteins than in steam-injected formula, reflecting the more severe thermal process of in-can sterilization. In vitro and in vivo digestion assays indicated that steam injection improved protein digestibility, supposedly by denaturation, while the improvement seemed to be overwhelmed by formation of aggregates that showed resistance to digestion in in-can sterilized formula. Adverse effects of heat treatment on protein digestibility are more likely to be manifested in milk-based formulas than in cow's milk. Although the differences might be of limited significance in terms of amino acid bioavailability, these results emphasize the importance of protein quality of raw materials and selection of heating processes. PMID:26161498

  6. Kinetics of Hydrolyzing Isolated Soy Protein by an Endopeptidase and its Conceptual Application in Process Engineering

    Directory of Open Access Journals (Sweden)

    Zebin Wang

    2012-04-01

    Full Text Available A response study and the effects of different parameters (pH, temperature and enzyme dose on kinetics of isolated soy protein hydrolysis by a trypsin-like endopeptidase (TL1 were conducted. Degree of hydrolysis (%DH data varied at different times under different hydrolysis conditions. Fitting the kinetics data to Michaelis-Menten kinetics model did not result in reasonable kinetic parameters, which implied that Michaelis-Menten kinetics was invalid for such a hydrolysis process. A kinetics model proposed by (Gonzalez-Tello, Camacho, Jurado, Paez, & Guadix, 1994 was found to fit the kinetics curve well and resulted in acceptable model parameters. A simple simulation example was performed to demonstrate the concept of how the kinetics equation could be applied in process engineering.

  7. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  8. Soy protein recovery in a solvent-free process using continuous liquid-solid circulating fluidized bed ion exchanger.

    Science.gov (United States)

    Prince, Andrew; Bassi, Amarjeet S; Haas, Christine; Zhu, Jesse X; Dawe, Jennifer

    2012-01-01

    Soy protein concentrates and soy protein isolates act as ingredients in bakery, meat and dairy products, baby formulas, starting materials for spun textured vegetable products, and other nutritional supplements. In this study, the effectiveness of a liquid-solid circulating fluidized bed (LSCFB) ion exchanger is demonstrated for the recovery of soluble soy proteins from full fat and defatted soy flour. Under steady-state operating conditions, about 50% of the proteins could be recovered from the feed streams entering the ion exchanger. The LSCFB was shown to be a promising system for the recovery of soy protein from both defatted and full fat soy flour solutions. As the ion exchange process captures dissolved proteins, the system may offer a less damaging form of processing compared with the acid precipitation process where soy protein aggregates form and functionality is affected. In addition, the LSCFB allows simultaneous adsorption and desorption of the proteins allowing for a continuous operation. No prefiltration of feed containing suspended particles is required as well, because fluidization is used in place of packed bed technology to improve on current ion exchange processes. PMID:22002948

  9. Whey protein processing influences formula-induced gut maturation in preterm pigs.

    Science.gov (United States)

    Li, Yanqi; Østergaard, Mette V; Jiang, Pingping; Chatterton, Dereck E W; Thymann, Thomas; Kvistgaard, Anne S; Sangild, Per T

    2013-12-01

    Immaturity of the gut predisposes preterm infants to nutritional challenges potentially leading to clinical complications such as necrotizing enterocolitis. Feeding milk formulas is associated with greater risk than fresh colostrum or milk, probably due to loss of bioactive proteins (e.g., immunoglobulins, lactoferrin, insulin-like growth factor, transforming growth factor-β) during industrial processing (e.g., pasteurization, filtration, spray-drying). We hypothesized that the processing method for whey protein concentrate (WPC) would affect gut maturation in formula-fed preterm pigs used as a model for preterm infants. Fifty-five caesarean-delivered preterm pigs were distributed into 4 groups given 1 of 4 isoenergetic diets: formula containing conventional WPC (filtration, multi-pasteurization, standard spray-drying) (CF); formula containing gently treated WPC (reduced filtration and pasteurization, gentle spray-drying) (GF); formula containing minimally treated WPC (rennet precipitation, reduced filtration, heat treatment colostrum (used as a positive reference group) (BC). Relative to CF, GF, and MF pigs, BC pigs had greater villus heights, lactose digestion, and absorption and lower gut permeability (P < 0.05). MF and BC pigs had greater plasma citrulline concentrations than CF and GF pigs and intestinal interleukin-8 was lower in BC pigs than in the other groups (P < 0.05). MF pigs had lower concentrations of intestinal claudin-4, cleaved caspase-3, and phosphorylated c-Jun than CF pigs (P < 0.05). The conventional and gently treated WPCs had similar efficacy in stimulating proliferation of porcine intestinal epithelial cells. We conclude that processing of WPC affects intestinal structure, function, and integrity when included in formulas for preterm pigs. Optimization of WPC processing technology may be important to preserve the bioactivity and nutritional value of formulas for sensitive newborns. PMID:24047702

  10. Effect of thermal processing on estimated metabolizable protein supply to dairy cattle from camelina seeds: relationship with protein molecular structural changes.

    Science.gov (United States)

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

    2014-08-20

    This study evaluated the effect of thermal processing on the estimated metabolizable protein (MP) supply to dairy cattle from camelina seeds (Camelina sativa L. Crantz) and determined the relationship between heat-induced changes in protein molecular structural characteristics and the MP supply. Seeds from two camelina varieties were sampled in two consecutive years and were either kept raw or were heated in an autoclave (moist heating) or in an air-draft oven (dry heating) at 120 °C for 1 h. The MP supply to dairy cattle was modeled by three commonly used protein evaluation systems. The protein molecular structures were analyzed by Fourier transform/infrared-attenuated total reflectance molecular spectroscopy. The results showed that both the dry and moist heating increased the contents of truly absorbable rumen-undegraded protein (ARUP) and total MP and decreased the degraded protein balance (DPB). However, the moist-heated camelina seeds had a significantly higher (P camelina seeds. The regression equations showed that intensities of the protein molecular structural bands can be used to estimate the contents of ARUP, MP, and DPB with high accuracy (R(2) > 0.70). These results show that protein molecular structural characteristics can be used to rapidly assess the MP supply to dairy cattle from raw and heat-treated camelina seeds. PMID:25046194

  11. Effects on cell growth processes (mitosis, synthesis of nucleic acids and of proteins). Chapter 7

    International Nuclear Information System (INIS)

    A review is presented of reports of the interference of -SH radioprotective agents with cell division and with the processes of nucleic acid and protein synthesis which are a prerequisite for mitosis. Mitotic activity is inhibited to the same extent in mammalian tissues as in cultures of animal and plant cells and bacteria. With cultured cells, the toxicity and the antimitotic activity have been found to be at their highest level for intermediate concentrations of the compound and to decrease for higher and lower concentrations. Inhibition of the synthesis of nucleic acids by -SH radioprotective substances has been observed with cultures of cells and bacteria and in mammalian tissues. In vitro interactions with the structures of free DNA and nucleoprotein have also been studied. The extent to which such complexes between the protective agent and DNA or nucleoprotein occur in vivo is not known. A depression of protein synthesis has been observed, and participates in the more general inhibition of growth processes. Possible mechanisms of these effects are discussed. (U.K.)

  12. Effects of sonication and high-pressure carbon dioxide processing on enzymatic hydrolysis of egg white proteins

    OpenAIRE

    Knežević-Jugović Zorica D.; Stefanović Andrea B.; Žuža Milena G.; Milovanović Stoja L.; Jakovetić Sonja M.; Manojlović Verica B.; Bugarski Branko M.

    2012-01-01

    The objectives of this study were to examine the effect of sonication and high-pressure carbon dioxide processing on proteolytic hydrolysis of egg white proteins and antioxidant activity of the obtained hydrolysates. It appeared that the ultrasound pretreatment resulted in an increase in the degree of hydrolysis of the enzymatic reaction while the high-pressure carbon dioxide processing showed an inhibition effect on the enzymatic hydrolysis of egg white proteins to some extent. The ant...

  13. Effect of extrusion processing on immune activation properties of hazelnut protein in a mouse model.

    Science.gov (United States)

    Ortiz, Tina; Para, Radhakrishna; Gonipeta, Babu; Reitmeyer, Mike; He, Yingli; Srkalovic, Ines; Ng, Perry K W; Gangur, Venu

    2016-09-01

    Although food processing can alter food allergenicity, the impact of extrusion processing on in vivo hazelnut allergenicity is unknown. Here, we tested the hypothesis that extrusion processing will alter the immune activation properties of hazelnut protein (HNP) in mice. Soluble extrusion-processed HNP (EHNP) was prepared and evaluated for immune response using an established transdermal sensitization mouse model. Mice were sensitized with identical amounts of EHNP versus raw HNP. After confirming systemic IgE, IgG1 and IgG2a antibody responses, oral hypersensitivity reaction was quantified by hypothermia shock response (HSR). Mechanism was studied by measuring mucosal mast cell (MMC) degranulation. Compared to raw HNP, the EHNP elicited slower but similar IgE antibody (Ab) response, lower IgG1 but higher IgG2a Ab response. The EHNP exhibited significantly lower oral HSR as well as MMC degranulation capacity. These results demonstrate that the extrusion technology can be used to produce soluble HNP with altered immune activation properties. PMID:27251648

  14. Novel Protein-Protein Contacts Facilitate mRNA 3'-Processing Signal Recognition by Rna15 and Hrp1.

    Energy Technology Data Exchange (ETDEWEB)

    Leeper, Thomas C; Qu, Xiangping; Lu, Connie; Moore, Claire; Varani, Gabriele

    2010-06-19

    Precise 3'-end processing of mRNA is essential for correct gene expression, yet in yeast, 3'-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3'-processing and are recognized by the RNAbinding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein–protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3'-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.

  15. Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

    Institute of Scientific and Technical Information of China (English)

    Olena; V; Bogdanova; Larysa; I; Kot; Kateryna; V; Lavrova; Volodymyr; B; Bogdanov; Erica; K; Sloan; Tetyana; V; Beregova; Ludmyla; I; Ostapchenko

    2010-01-01

    AIM:To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration. METHODS:Gastric lesions were induced in rats using restraint cold stress.To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair,total activity of protein tyrosine kinase(PTK),protein tyrosine phosphatase (PTP),antioxidant enzymes,nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase,hydroxyl radical and zinc levels were assayed in parallel. RESULTS:Ulcer provocation induced an immediate decrease in tyrosine kinase(40% in plasma membranes and 56% in cytosol,(P<0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol),followed by 2.3-2.4-fold decrease (P<0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity,30% increase (P<0.05) in catalase activity,2.3-fold inhibition (P<0.05) of glutathione peroxidase,3.3-fold increase (P<0.05) in hydroxyl radical content,and 2.3-fold decrease (P<0.05) in zinc level in gastric mucosa.NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration,PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase,P<0.05),but remained inhibited(1.6-3-fold decrease on days 3,4 and 5,P<0.05) in the cytosol.Tyrosine phosphatases remained inhibited both in membranes and cytosol(1.5-2.4-fold,P< 0.05).NOS activity remained increased on days 1,2 and 3(3.8-,2.6-,2.2-fold,respectively,P<0.05).Activity of SOD increased 1.6 times(P<0.05)days 4 and 5 after stress.Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3-and 2-fold,respectively,P<0.05)on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P<0.05) at the beginning,and 1.6-2.3-fold (P<0.05) during ulcer recuperation

  16. Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

    Directory of Open Access Journals (Sweden)

    Olena V Bogdanova

    2010-11-01

    Full Text Available AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration.METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK, protein tyrosine phosphatase (PTP, antioxidant enzymes, nitric oxide synthase (NOS, 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel.RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05 and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol, followed by 2.3-2.4-fold decrease (P < 0.05 in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD activity, 30% increase (P < 0.05 in catalase activity, 2.3-fold inhibition (P < 0.05 of glutathione peroxidase, 3.3-fold increase (P < 0.05 in hydroxyl radical content, and 2.3-fold decrease (P < 0.05 in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05, but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05 in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05. NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05. Activity of SOD increased 1.6 times (P < 0.05 days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05 on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05 at the

  17. Independent relationship between amyloid precursor protein (APP dimerization and γ-secretase processivity.

    Directory of Open Access Journals (Sweden)

    Joo In Jung

    Full Text Available Altered production of β-amyloid (Aβ from the amyloid precursor protein (APP is closely associated with Alzheimer's disease (AD. APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99 impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43. Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD. This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.

  18. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    DEFF Research Database (Denmark)

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane;

    2016-01-01

    replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear...... whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the...

  19. The promoter of filamentation (POF1 protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

    Directory of Open Access Journals (Sweden)

    Costa Iris M

    2011-12-01

    Full Text Available Abstract Background The gene YCL047C, which has been renamed promoter of filamentation gene (POF1, has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD. Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme in vivo. Conclusions Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

  20. Production process for high-quality pea-protein isolate with low content of oligosaccharides and phytate.

    Science.gov (United States)

    Fredrikson, M; Biot, P; Alminger, M L; Carlsson, N G; Sandberg, A S

    2001-03-01

    A process for pea-protein isolate production, resulting in low content of phytate and oligosaccharides, has been developed. Oligosaccharides were removed from the protein fraction through ultrafiltration. Ultrafiltration of 50- and 100-kD molecular-weight cutoffs (MWCOs) were tested, and both effectively separated the oligosaccharides from the protein. Phytate degradation was achieved by incubation of the pea-protein solution by addition of exogenous phytase enzyme. An almost complete degradation of inositol hexa-, penta-, tetra-, and triphosphates was reached using an incubation time of 1 h. The reduced content of oligosaccharides and inositol phosphates is likely to result in reduced flatulence and improved mineral bioavailability. These qualities of the pea-protein isolate make it a suitable protein source for infant formula production. PMID:11312837

  1. Bioconversion of Radiation Processed Dried Tomato Pomace to High Protein Animal Fee

    International Nuclear Information System (INIS)

    The increasing expansion of agro-industrial activity over the last 50 years has led to the accumulation of a large quantity of organic residues all over the world that they have become a threat to the environment. Bioconversion of these wastes seems to be a practical and promising alternative for increasing their nutritional value, transforming them into animal feed and thus producing a value added product. Radiation processing has the capability to reduce or eliminate pathogenic bacteria, insects and parasites, thereby increasing the utilization and sustainable management of waste organic matter from food production and processing while contributing to improve food quality and reducing the environmental impact of the wastes. The main purpose of this study was to evaluate the effect of radiation treatment at 25 kGy and fermentation process by Aspergillus niger, on crude and soluble protein, amino acid profile, available lysine and in vitro digestibility of dried tomato pomace (DTP), the by-product of the tomato canning industry. The study has also, investigated the effect of supplementation of 30% of raw or processed DTP meal in food of male Albino rats for six weeks on body and liver weight evaluation and the effect on blood lipid pattern. The work concluded that the combination between the irradiation of DTP at 25 kGy and fermentation process has increased the nutritional value of treated DTP meal and improved the plasma and liver lipid pattern of rats. Therefore, the combination treatment has beneficial effects on recycling of DTP and permits it to be included in monogastric animals' food without any health hazard or nutritional problem

  2. Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk.

    Science.gov (United States)

    Zhao, Jianguo; Xu, Weijie; Ross, Jason W; Walters, Eric M; Butler, Stephen P; Whyte, Jeff J; Kelso, Lindsey; Fatemi, Mostafa; Vanderslice, Nicholas C; Giroux, Keith; Spate, Lee D; Samuel, Melissa S; Murphy, Cliff N; Wells, Kevin D; Masiello, Nick C; Prather, Randall S; Velander, William H

    2015-01-01

    Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to <20% of the world's population. We used transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock. PMID:26387706

  3. [Study of the process of thermal aggregation of several representative tobamovirus coat proteins].

    Science.gov (United States)

    Abu-Eid, M; Kust, S V; Makeeva, I V; Novikov, V K; Dobrov, E N

    1994-01-01

    The role of the specific region of the tobacco mosaic virus (TMV) coat protein (CP) molecule (called "70A degree-region") in the regulation of ordered and unordered CP aggregation was investigated. CPs of the wild type TMV (strain U1), of temperature sensitive mutant with two amino acid substitutions in the "70A degree-region", and of cucumber virus 3 which is related to TMV but has a completely different structure in the "70A degree-region" were used. With the help of two different tests the processes of temperature-induced unordered aggregation of these three CPs were compared in solutions of different ionic strength and pH. On the basis of the data obtained it was concluded that the "70A-region" represents the most thermolabile region in the TMV CP molecule and that local thermal denaturation of this region results in unordered aggregation, when solution conditions (ionic strength and pH) favor formation of relatively large ordered aggregates (20S-"disks" or helical repolymerized protein). PMID:8065382

  4. Incorporation of radiolabeled whey proteins into casein micelles by heat processing

    International Nuclear Information System (INIS)

    Skim milk was heated at .70, 95, and 140 degree C to simulate the processes of pasteurization, forewarming, and UHT sterilization, and the specific interactions between α-lactalbumin or β-lactoglobulin and the caseins studied using tracer amounts of added 14C-labeled whey protein. Radioactivities of the whey and of the washed casein pellets from renneted skim milk were measured and the extent of the interaction estimated. Upon heating skim milk at 70 degree C for 45 s, less than 2% β-lactoglobulin and less than .3% α-lactalbumin were incorporated into the curd. Heating at 95 degree C for .5 to 20 min resulted in 58 to 85% of the β-lactoglobulin and 8 to 55% of the α-lactalbumin becoming associated with the curd. Heating at 140 degree C for 2 and 4 s caused 43 and 54% of the β-lactoglobulin and 9 and 12% of the α-lactalbumin, respectively, to be bound to the curd fraction. The radiolabeling technique is very sensitive and useful for tracing low levels of interaction between whey proteins and casein in heated milk systems

  5. Use of fish processing waste as protein source in diet for Nile tilapia (Orechromis niloticus

    Directory of Open Access Journals (Sweden)

    Chotipuntu, P.

    2005-02-01

    Full Text Available Five diets were prepared using fish processing waste meal (FMFP to replace fish meal (FM at inclusion levels of 0, 25, 50, 75 and 100%. Frog diet was used as a control diet. Nile tilapia (Oreochromis niloticus were reared in laboratory conditions for 8 weeks. It was found that substitution levels of protein from FMFP in the tested diets reduced growth and feed efficiency of tilapia (p<0.05. However, the differences looks like significant trend especially that between the 100% substitution level and the frog diet. Substitution of FM by FMFD at 75% reduced cost of feed by 15.35%. It was concluded that up to 75% inclusion of FMFD in the diet of tilapia could support normal growth of Nile tilapia with the potential for substitution of FM.

  6. Pulsed laser deposition of the lysozyme protein: an unexpected “Inverse MAPLE” process

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin; Tabetah, Marshall; Zhigilei, Leonid V.; Dinescu, Maria

    2012-01-01

    material directly for film production, as in PLD (pulsed laser deposition), where the film molecules may undergo strong fragmentation. In this presentation we report an alternative surprising mechanism for film deposition of the protein lysozyme in vacuum, when a small amount of residual water drives the...... which is used in food processing and is also an important constituent of human secretions such as sweat and saliva. It has a well-defined mass (14307 u) and can easily be detected by mass spectrometric methods such as MALDI (Matrix-assisted laser desorption ionization) in contrast to many other organic...... possible. The experimental results are explained with the help of molecular-level computer simulations. The simulations show that pure lysozyme cannot ablate without complete fragmentation. However, small pockets of trapped water provide the necessary expansion of the target and the ejection of intact...

  7. Formaldehyde treatment of proteins can constrain presentation to T cells by limiting antigen processing.

    OpenAIRE

    Di Tommaso, A; De Magistris, M T; Bugnoli, M.; Marsili, I; Rappuoli, R; Abrignani, S.

    1994-01-01

    Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (p...

  8. Validation Processes of Protein Biomarkers in Serum—A Cross Platform Comparison

    Directory of Open Access Journals (Sweden)

    Harald Seitz

    2012-09-01

    Full Text Available Due to insufficient biomarker validation and poor performances in diagnostic assays, the candidate biomarker verification process has to be improved. Multi-analyte immunoassays are the tool of choice for the identification and detailed validation of protein biomarkers in serum. The process of identification and validation of serum biomarkers, as well as their implementation in diagnostic routine requires an application of independent immunoassay platforms with the possibility of high-throughput. This review will focus on three main multi-analyte immunoassay platforms: planar microarrays, multiplex bead systems and, array-based surface plasmon resonance (SPR chips. Recent developments of each platform will be discussed for application in clinical proteomics, principles, detection methods, and performance strength. The requirements for specific surface functionalization of assay platforms are continuously increasing. The reasons for this increase is the demand for highly sensitive assays, as well as the reduction of non-specific adsorption from complex samples, and with it high signal-to-noise-ratios. To achieve this, different support materials were adapted to the immobilized biomarker/ligand, allowing a high binding capacity and immobilization efficiency. In the case of immunoassays, the immobilized ligands are proteins, antibodies or peptides, which exhibit a diversity of chemical properties (acidic/alkaline; hydrophobic/hydrophilic; secondary or tertiary structure/linear. Consequently it is more challenging to develop immobilization strategies necessary to ensure a homogenous covered surface and reliable assay in comparison to DNA immobilization. New developments concerning material support for each platform are discussed especially with regard to increase the immobilization efficiency and reducing the non-specific adsorption from complex samples like serum and cell lysates.

  9. Effect of two-step functionalization of Ti by chemical processes on protein adsorption

    Science.gov (United States)

    Pisarek, M.; Roguska, A.; Andrzejczuk, M.; Marcon, L.; Szunerits, S.; Lewandowska, M.; Janik-Czachor, M.

    2011-07-01

    Titanium and its alloys are widely used for orthopedic and dental implants because of their superior mechanical properties, low modulus, excellent corrosion resistance and good biocompatibility. However, it takes several months for titanium implants and bone tissue to reach integration. Hence, there is growing interest in shortening the process of osseointegration and thereby reducing surgical restrictions. Various surface modifications have been applied to form a bioactive titanium oxide layer on the metal surface, which is known to accelerate osseointegration. The present work shows that titanium dioxide (TiO 2) layers formed on titanium substrates by etching in a solution of sodium hydroxide (NaOH) or hydrogen peroxide/phosphoric acid (H 3PO 4/H 2O 2, with a volume ratio of 1:1) are highly suitable pre-treatments for apatite-like coating deposition. Using a two-step procedure (etching in an alkaline or acidic solution followed by soaking in Hanks' medium), biomimetic calcium phosphate coatings were deposited on porous TiO 2 layers. The combined effects of surface topography and chemistry on the formation of the calcium phosphate layer are presented. The topography of the TiO 2 layers was characterized using HR-SEM and AFM techniques. The nucleation and growth of calcium phosphate (Ca-P) coatings deposited on TiO 2 porous layers from Hanks' solution was investigated using HR-SEM microscopy. AES, XPS and FTIR surface analytical techniques were used to characterize the titanium dioxide layers before and after deposition of the calcium phosphate coatings, as well as after the process of protein adsorption. To evaluate the potential use of such materials for biomedical applications, the adsorption of serum albumin, the most abundant protein in the blood, was studied on such surfaces.

  10. Inhibition of ABC transport proteins by oil sands process affected water.

    Science.gov (United States)

    Alharbi, Hattan A; Saunders, David M V; Al-Mousa, Ahmed; Alcorn, Jane; Pereira, Alberto S; Martin, Jonathan W; Giesy, John P; Wiseman, Steve B

    2016-01-01

    The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI(+) and ESI(-) mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O(+), SO(+), and NO(+) chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2(-)), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates

  11. Role of protein phosphorylation in the regulation of cell cycle and DNA-related processes in bacteria

    Directory of Open Access Journals (Sweden)

    Transito eGarcia-Garcia

    2016-02-01

    Full Text Available In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes.

  12. Optimization of the protein concentration process from residual peanut oil-cake

    Directory of Open Access Journals (Sweden)

    Gayol, M. F.

    2013-12-01

    Full Text Available The objective of this study was to find the best process conditions for preparing protein concentrate from residual peanut oil-cake (POC. The study was carried out on POC from industrial peanut oil extraction. Different protein extraction and precipitation conditions were used: water/ flour ratio (10:1, 20:1 and 30:1, pH (8, 9 and 10, NaCl concentration (0 and 0.5 M, extraction time (30, 60 and 120 min, temperature (25, 40 and 60 °C, extraction stages (1, 2 and 3, and precipitation pH (4, 4.5 and 5. The extraction and precipitation conditions which showed the highest protein yield were 10:1 water/flour ratio, extraction at pH 9, no NaCl, 2 extraction stages of 30 min at 40 °C and precipitation at pH 4.5. Under these conditions, the peanut protein concentrate (PC contained 86.22% protein, while the initial POC had 38.04% . POC is an alternative source of protein that can be used for human consumption or animal nutrition. Therefore, it adds value to an industry residue.El objetivo de este trabajo fue encontrar las mejores condiciones para obtener un concentrado de proteínas a partir de la torta residual de maní (POC. El estudio se llevó a cabo en POC provenientes de la extracción industrial de aceite de maní. Se utilizaron distintas condiciones para la extracción y precipitación de proteínas: relación agua / harina (10:1, 20:1 y 30:1, pH de extracción (8, 9 y 10, concentración de NaCl (0 y 0,5 M, tiempo de extracción (30, 60 y 120 min, temperatura (25, 40 y 60 °C, número de etapas de extracción (1, 2 y 3, y el pH de precipitación (4, 4,5 y 5. Las condiciones de extracción y de precipitación que mostraron mayor rendimiento de proteína fueron: relación de 10:1 en agua / harina, pH de extracción de 9, en ausencia de NaCl, 2 etapas de extracción de 30 min cada una a 40 °C y el pH de precipitación de 4,5. En estas condiciones, el concentrado de proteína de maní (PC fue de 86,22%, mientras que el porcentaje de proteínas de

  13. Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein

    Directory of Open Access Journals (Sweden)

    Saoussen Ben Halima

    2016-03-01

    Full Text Available Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD. AD is characterized by toxic β-amyloid (Aβ peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP. β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects.

  14. Inter-laboratory validation study of two immunochemical methods for detection of processed ruminant proteins.

    Science.gov (United States)

    van Raamsdonk, L W D; Margry, R J C F; van Kaathoven, R G C; Bremer, M G E G

    2015-10-15

    In order to facilitate safe re-introduction of non-ruminant processed animal proteins (PAPs) in aqua feed, two immunoassays have been tested in an interlaboratory study for their capability to detect ruminant PAPs processed under European conditions. The sensitivity of the MELISA-TEK assay was improved by applying a specific extraction kit. Six approved blank pork and poultry samples were adulterated to produce 15 samples spiked at 0.5%, 1.0% and 2.0% with ruminant material, sterilised at either 133 °C or 137 °C. Fourteen participants investigated the 6 blanks and 15 spiked samples, making 21 samples for the final test. For both assays specificity and sensitivity were at 97% or higher. Concordance and accordance were higher than 95% with one exception. The results indicate that both assays provided correct results at 0.5% and higher for the detecting ruminant PAPs (sterilised at 133 °C) in non-ruminant PAPs. Given the 2% upper limit of ruminant PAPs in non-ruminant PAPs for avoiding an increase in BSE incidents, these methods are fit for monitoring non-ruminant PAPs intended for aqua feed. PMID:25952876

  15. Engineered G-protein coupled receptors are powerful tools to investigate biological processes and behaviors

    Directory of Open Access Journals (Sweden)

    Charles D Nichols

    2009-10-01

    Full Text Available Understanding how discreet tissues and neuronal circuits function in relation to the whole organism to regulate physiological processes and behaviors is a fundamental goal of modern biological science. Powerful and important new tools in this discovery process are modified G-protein coupled receptors (GPCRs known as ‘Receptors Activated Solely by Synthetic Ligands (RASSLs,’ and ‘Designer Receptors Exclusively Activated by a Designer Drug (DREADDs.’ Collectively, these are GPCRs modified either through rational design or directed molecular evolution, that do not respond to native ligand, but functionally respond only to synthetic ligands. Importantly, the utility of these receptors is not limited to examination of the role of GPCR-coupled effector signal transduction pathways. Due to the near ubiquitous expression of GPCRs throughout an organism, this technology, combined with whole animal transgenics to selectively target expression, has the ability to regulate activity of discreet tissues and neuronal circuits through effector pathway modulation to study function and behavior throughout the organism. Advantages over other systems currently used to modify in vivo function include the ability to rapidly, selectively and reversibly manipulate defined signal transduction pathways both in short term and long term studies, and no need for specialized equipment due to convenient systemic treatment with activating ligand.

  16. A magnetic adsorbent-based process for semi-continuous PEGylation of proteins

    DEFF Research Database (Denmark)

    Ottow, Kim Ekelund; Maury, Trine Lütken; Hobley, Timothy John;

    2011-01-01

    minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed......A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to...... into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and...

  17. A process efficiency assessment of serum protein removal from milk using ceramic graded permeability microfiltration membrane.

    Science.gov (United States)

    Tremblay-Marchand, D; Doyen, A; Britten, M; Pouliot, Y

    2016-07-01

    Microfiltration (MF) is a well-known process that can be used in the dairy industry to separate caseins from serum proteins (SP) in skim milk using membranes with a pore diameter of 0.1μm. Graded permeability ceramic membranes have been studied widely as means of improving milk fractionation by overcoming problems encountered with other MF membranes. The ideal operating parameters for process efficiency in terms of membrane selectivity, permeate flux, casein loss, SP transmission, energy consumption, and dilution with water remain to be determined for this membrane. Our objective was to evaluate the effects of transmembrane pressure (TMP), volumetric concentration factor (VCF), and diafiltration on overall process efficiency. Skim milk was processed using a pilot-scale MF system equipped with 0.72-m(2) graded permeability membranes with a pore size of 0.1μm. In the first experiment, in full recycle mode, TMP was set at 124, 152, 179, or 207 kPa by adjusting the permeate pressure at the outlet. Whereas TMP had no significant effect on permeate and retentate composition, 152 kPa was found to be optimal for SP removal during concentration and concentration or diafiltration experiments. When VCF was increased to 3×, SP rejection coefficient increased along with energy consumption and total casein loss, whereas SP removal rate decreased. Diafiltering twice allowed an increase in total SP removal but resulted in a substantial increase in energy consumption and casein loss. It also reduced the SP removal rate by diluting permeate. The membrane surface area required for producing cheese milk by blending whole milk, cream, and MF retentate (at different VCF) was estimated for different cheese milk casein concentrations. For a given casein concentration, the same quantity of permeate and SP would be produced, but less membrane surface area would be needed at a lower retentate VCF. Microfiltration has great potential as a process of adding value to conventional

  18. Effects of sonication and high-pressure carbon dioxide processing on enzymatic hydrolysis of egg white proteins

    Directory of Open Access Journals (Sweden)

    Knežević-Jugović Zorica D.

    2012-01-01

    Full Text Available The objectives of this study were to examine the effect of sonication and high-pressure carbon dioxide processing on proteolytic hydrolysis of egg white proteins and antioxidant activity of the obtained hydrolysates. It appeared that the ultrasound pretreatment resulted in an increase in the degree of hydrolysis of the enzymatic reaction while the high-pressure carbon dioxide processing showed an inhibition effect on the enzymatic hydrolysis of egg white proteins to some extent. The antioxidant activity of the obtained hydrolysates was improved by ultrasound pretreatment of egg white proteins at the pH 8.3. Thus, the combination of ultrasound pretreatment at the pH 8.3 and subsequent enzymatic hydrolysis with alcalase at 50°C and pH 8.0 could offer a new approach to the improvement of the functional properties of egg white proteins and their biological activity. [Projekat Ministarstva nauke Republike Srbije, br. E!6750

  19. Reexpression of glial fibrillary acidic protein rescues the ability of astrocytoma cells to form processes in response to neurons

    OpenAIRE

    1994-01-01

    Astroglial cells play an important role in orchestrating the migration and positioning of neurons during central nervous system development. Primary astroglia, as well as astrocytoma cells will extend long stable processes when co-cultured with granule neurons. In order to determine the function of the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astroglia and astrocytoma cells, we suppressed the expression of GFAP by stable transfection of an anti- sense...

  20. Enzymatic Hydrolysis of Salmon By-products: Effect of Process Conditions on ACE Inhibiting Activities of Fish Protein Hydrolysates

    OpenAIRE

    Five, Kathrine

    2013-01-01

    By-products from the salmon farming industry contain valuable components, such as proteins and lipids. By-products like frames, heads and viscera can be used as raw material for the production of fish protein hydrolysates with high nutritional value, but also bioactive properties. The hydrolysates are produced by enzymatic hydrolysis using endogenous and commercial enzymes, and the process conditions and raw material influence the properties of the hydrolysate. The first aim of this thesis wa...

  1. Oxidation of lipid and protein in horse mackerel (Trachurus trachurus) mince and washed minces during processing and storage

    DEFF Research Database (Denmark)

    Eymard, Sylvie; Baron, Caroline; Jacobsen, Charlotte

    2009-01-01

    Protein and lipid oxidation was followed during processing and storage of mince and washed minces prepared from horse mackerel (Trachurus trachunts). Briefly horse mackerel mince (MO) was washed with three volumes of water, mimicking the surimi production and different washed products were obtained......: M1, M2 and M3, with one, two and three washing steps, respectively. The different products were characterised (i.e. lipid content, protein, water, iron, fatty acid profile and tocopherol content) and analysed for protein and lipid oxidation in order to investigate the impact of the washing steps on...

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

    OpenAIRE

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

    2014-01-01

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

  3. Characterization of flows in micro contractions using micro PIV and CFD to study the protein aggregation process

    Science.gov (United States)

    Tovar-Lopez, Francisco J.; Mitchell, Arnan; Rosengarten, Gary

    2007-12-01

    Protein aggregation is arguably the most common and troubling manifestation of protein instability, encountered in almost all stages of protein drug development. The production process in the pharmaceutical industry can induce flows with shear and extensional components and high strain rates which can affect the stability of proteins. We use a microfluidic platform to produce accurately controlled strain regions in order to systematically study the main parameters of the flow involved in the protein aggregation. This work presents a characterization of the pressure driven flow encountered in arrays of micro channels. The micro channels were fabricated in polydimethyl siloxane (PDMS) using standard soft-lithography techniques with a photolithographically patterned KMPR mold. We present a relationship of the main geometrical variables of the micro channels and its impact on the extensional strain rate along the center line, for different cross sectional shapes and over a range of strain rates typically encountered in protein processing. Computational Fluid Dynamics (CFD) simulations have been carried out to gain more detailed local flow information, and the results have been validated with experiments. We show good agreement between the CFD and experiments and demonstrate the use of microfluidics in the production of a large range of controllable shear and extensional rates that can mimic large scale processing conditions.

  4. Integration of a central protein repository into a standard data processing application for mining proteomics data

    DEFF Research Database (Denmark)

    Fritzemeier, Kai; Kristensen, Jakob; Larsen, Martin Røssel;

    time consuming and at times nearly impossible task to distinguish known proteins from novel proteins in these data sets without proper annotation and comparison with literature sources. Tools are needed that can handle the complexity of these data including: redundancy (same protein but different...... accession codes), different protein database accession codes or outdated accession codes and protein annotation. To resolve these issues we have developed a consolidated proteomics database providing annotations to Proteome Discoverer via direct integrated web service technology – a repository that enables...

  5. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (∼7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. PMID:26627201

  6. Synthesis of gold and silver nanoparticle S-ovalbumin protein conjugates by in situ conjugation process

    Energy Technology Data Exchange (ETDEWEB)

    Joshi, Deepti, E-mail: deeptimishrajoshi@gmail.com; Soni, R. K. [Indian Institute of Technology Delhi, Physics Department (India)

    2015-05-15

    Pure gold and silver nanoparticle (NP) generation and their conjugation with protein S-ovalbumin using in situ conjugation process have been reported. The in situ conjugation involves nanosecond pulse laser ablation of pure metal target in the protein S-ovalbumin solution. Transmission electron microscopy (TEM) and UV–Visible absorption results show decrease in mean NP size along with narrow particle size distribution on ablation in S-ovalbumin solution as compared to ablation in water for both Au and Ag NPs. Also, the NP size reduction was found to be dependent on the concentration of S-ovalbumin. For AuNPs, spherical NPs of mean size 4 nm with particle size distribution 2–6 nm were obtained at 300 nM S-ovalbumin concentration. Further, it has been observed that the resultant in situ-conjugated colloid gold and silver NP solutions were quite stable even in the presence of NaCl at physiological salt concentration (0.15 M). On post-laser irradiation (532 nm, 15 mJ) for 20 min, 9 nm red shift in surface plasmon resonance peak (SPR), along with increased broadening towards longer wavelength, was observed in the AuNPs–S-ovalbumin sample. Further increase in the time of irradiation showed shift in AuNPs–S-ovalbumin SPR towards lower wavelength. On laser irradiation (532 nm, 15 mJ) for 20 min, no significant change was observed in the line shape of the plasmon absorption band of the AgNPs–S-ovalbumin conjugate. FTIR spectra revealed that S-ovalbumin peptide backbone and secondary structure remain unchanged on laser irradiation during in situ conjugation process. Thus, integrity of S-ovalbumin does not get affected, and no degradation of S-ovalbumin takes place on laser-induced in situ conjugation. Raman results confirm that both Au and Ag NPs interact with S-ovalbumin via thiol-bearing cysteine residues of the disulfide bond.

  7. Synthesis of gold and silver nanoparticle S-ovalbumin protein conjugates by in situ conjugation process

    International Nuclear Information System (INIS)

    Pure gold and silver nanoparticle (NP) generation and their conjugation with protein S-ovalbumin using in situ conjugation process have been reported. The in situ conjugation involves nanosecond pulse laser ablation of pure metal target in the protein S-ovalbumin solution. Transmission electron microscopy (TEM) and UV–Visible absorption results show decrease in mean NP size along with narrow particle size distribution on ablation in S-ovalbumin solution as compared to ablation in water for both Au and Ag NPs. Also, the NP size reduction was found to be dependent on the concentration of S-ovalbumin. For AuNPs, spherical NPs of mean size 4 nm with particle size distribution 2–6 nm were obtained at 300 nM S-ovalbumin concentration. Further, it has been observed that the resultant in situ-conjugated colloid gold and silver NP solutions were quite stable even in the presence of NaCl at physiological salt concentration (0.15 M). On post-laser irradiation (532 nm, 15 mJ) for 20 min, 9 nm red shift in surface plasmon resonance peak (SPR), along with increased broadening towards longer wavelength, was observed in the AuNPs–S-ovalbumin sample. Further increase in the time of irradiation showed shift in AuNPs–S-ovalbumin SPR towards lower wavelength. On laser irradiation (532 nm, 15 mJ) for 20 min, no significant change was observed in the line shape of the plasmon absorption band of the AgNPs–S-ovalbumin conjugate. FTIR spectra revealed that S-ovalbumin peptide backbone and secondary structure remain unchanged on laser irradiation during in situ conjugation process. Thus, integrity of S-ovalbumin does not get affected, and no degradation of S-ovalbumin takes place on laser-induced in situ conjugation. Raman results confirm that both Au and Ag NPs interact with S-ovalbumin via thiol-bearing cysteine residues of the disulfide bond

  8. Advanced cleanup process of the free-flow microfluidic device for protein analysis

    International Nuclear Information System (INIS)

    The treatment of samples preparation is generally recognized as a bottleneck for the rapid analysis of protein because of the off-chip performance in many cases. In this study, we used the charge characteristics of protein to develop a simple and rapid electro-microfluidic desalting system as an effective means of cleaning up protein sample. When we loaded a urea-rich protein sample and a buffer solution into a free-flow zone electrophoresis (FFZE) chamber, the microfluidic device was able to separate the charged protein sample and the non-charged urea. With a 90 V electric field in the FFZE chamber, the removal efficiency of the urea was about 88% and the recovery of the protein was 78%. In addition, the desalted protein sample used in this device showed significant improvement with respect to the MALDI-TOF-MS spectrum signal of a fusion protein, which was fused to the gold-binding polypeptide with enhanced green fluorescent protein, as a model protein. The inflow of the purified fusion protein sample can be successfully immobilized on the gold surface and analyzed by confocal fluorescence microscopy and surface plasmon resonance for biotechnological sensors

  9. Searching the literature for proteins facilitates the identification of biological processes, if advanced methods of analysis are linked

    DEFF Research Database (Denmark)

    Bauer, Johann; Bussen, Markus; Wise, Petra;

    2016-01-01

    exposed to microgravity conditions. RESEARCH DESIGN AND METHODS: We retrieved publications about microgravity related studies on each type of cell, extracted the proteins mentioned therein and analyzed them subsequently using Pathway Studio aiming to identify biological processes affected by microgravity...... of gravity is common to all the different cell types, at least some of the 19 biological processes could play a role in cellular adaption to microgravity. The application of computer programs, helping to extract and analyze proteins and genes mentioned in publications will become essential for scientists...

  10. Retromer Binds the FANSHY Sorting Motif in SorLA to Regulate Amyloid Precursor Protein Sorting and Processing

    DEFF Research Database (Denmark)

    Fjorback, Anja W; Seaman, Matthew; Gustafsen, Camilla;

    2012-01-01

    levels of retromer proteins are altered in AD. Here we report that sorLA and retromer functionally interact in neurons to control trafficking and amyloidogenic processing of APP. We have identified a sequence (FANSHY) in the cytoplasmic domain of sorLA that is recognized by the VPS26 subunit of the......sorLA is a sorting receptor for amyloid precursor protein (APP) genetically linked to Alzheimer's disease (AD). Retromer, an adaptor complex in the endosome-to-Golgi retrieval pathway, has been implicated in APP transport because retromer deficiency leads to aberrant APP sorting and processing and...

  11. A possible role of repair proteins BRCA1 and DNA-PK in the processing of oxidative DNA damage

    Directory of Open Access Journals (Sweden)

    Alexandros G Georgakilas

    2008-08-01

    Full Text Available BRCA1 and DNA-PK are two significant multifunctional proteins involved primarily in the processing of double strand breaks (DSBs. BRCA1 participates actively in homologous recombination (HR while DNA-PK in non-homologous end joining (NHEJ. In this mini review, we discuss all recent evidence for a possible involvement of these repair proteins also in the processing of oxidatively-induced DNA damage.Keyword: DNA damage, BRCA1, DNA-PKReceived: 6 June 2008, Accepted: 10 August 2008 Published online: 18 August 2008

  12. Potential application of electronic nose in processed animal proteins (PAP detection in feedstuffs

    Directory of Open Access Journals (Sweden)

    Dell'Orto V.

    2004-01-01

    Full Text Available Electronic nose and olfactometry techniques represent a modern analytical approach in food industry since they could potentially improve quality and safety of food processing. The aim of this study was to evaluate possible application of electronic nose in PA P detection and recognition in feed. For this purpose 6 reference feedstuffs (CRA-W / UE STRAT F E E D Project were used. The basis of the test samples was a compound feed for bovine fortified with processed animal proteins ( PAP consisting of meat and bone meal (MBM and/or fish meal at different concentrations. Each feed sample was tested in glass vials and the odour profile was determined by the ten MOS (metal oxide semi-conductor sensors of the electronic nose. Ten different descriptors, representing each ten sensors of electronic nose, were used to characterise the odour of each sample. In the present study, electronic nose was able to discriminate the blank sample from all other samples containing PA P ( M B M , fish meal or both. Samples containing either 0.5% of MBM or 5% of fish meal were identified, while samples containing a high fish meal content (5% associated with a low MBM content (0.5% were not discriminated from samples containing solely fish meal at that same high level (5%. This latter indicates that probably the high fish meal level, in samples containing both MBM and fish meal, tended to mask MBM odour. It was also evident that two odour descriptors were enough to explain 72.12% of total variability in odour pattern. In view of these results, it could be suggested that electronic nose and olfactometry techniques can provide an interesting approach for screening raw materials in feed industry, even though further studies using a wider set of samples are needed.

  13. Methods of detection, species identification and quantification of processed animal proteins in feedingstuffs

    Directory of Open Access Journals (Sweden)

    Fumière O.

    2009-01-01

    Full Text Available The ban of processed animal proteins (PAPs in feed for farmed animals led to a significant reduction of the number of bovine spongiform encephalopathy cases. Presently, optical microscopy remains the only reference method for the detection of PAPs to be applied for official control as required by Commission Directive 2003/126/EC. The legislation also foresees that other methods may be applied in addition to classical microscopy, if – for instance – they provide more information about the origin of the animal constituents. Therefore, alternative and complementary techniques were developed as such or in combination. The most promising ones seem to be PCR (Polymerase Chain Reaction, near infrared microscopy and imaging, as well as immunology. Within the framework of a PAP ban regardless of its species origin (total feed ban, most of the studies were mainly focused on the ability of the techniques to detect the presence of PAPs at 0.1% (mass percentage of constituents of animal origin in feed as indicated as limit of detection in the official method protocol. A possible modification of the legislation requires that the techniques are also able to determine their species origin and to quantify them. The present paper gives a state of the art of the different methods.

  14. Application of Protein Feed Processed by Microbial Fermentation to Dairy Cow

    Institute of Scientific and Technical Information of China (English)

    Sun Zhe; Liu Ying; Pan Hong-bao; Gao Xue-jun

    2014-01-01

    Methionine (Met) and lysine (Lys) have been reported as the first two limiting amino acids (AA) for maximum milk yield and milk protein production. Supplying these AA may improve microbial protein synthesis and therefore improve milk production without adding excess N to the environment. This observation utilized fermented soybean meal (SBM), cottonseed meal (CSM), rapeseed meal (RSM) and corn by Bacillus subtilis 168 and Leuconostoc mesenteroides as core feedstuffs to produce special biological protein feed for dairy cow. The results showed that the milk production, milk protein percentage, milk fat percentage and milk DM percentage of test groups in trial period were significantly more than those of the control group (P<0.01), the results showed that adding fermenting protein feed in dairy cow diets could significantly improve milk yield, milk protein and milk fat content. The economic benefits of actual application were analyzed, the group of 0.5%was the best compared with the other groups.

  15. A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization.

    Science.gov (United States)

    Qiu, Chen; McCann, Kathleen L; Wine, Robert N; Baserga, Susan J; Hall, Traci M Tanaka

    2014-12-30

    Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. Here we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an "L"-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Thus, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins. PMID:25512524

  16. Identification of an EF-Tu protein that is periplasm-associated and processed in Neisseria gonorrhoeae.

    Science.gov (United States)

    Porcella, S F; Belland, R J; Judd, R C

    1996-09-01

    A 44 kDa protein is a dominant component of periplasmic extracts of Neisseria gonorrhoeae. Peptide sequence generated from a cyanogen-bromide-cleaved fragment of this protein indicated sequence homology with elongation factor-Tu (EF-Tu). Polyclonal antiserum was made against the 44 kDa protein purified from periplasm extracts of N. gonorrhoeae. The preabsorbed antiserum was immunoblotted against whole-cell lysates on two-dimensional gels. A 44 kDa protein and a smaller 37 kDa protein were recognized by this antiserum. A N. gonorrhoeae gamma phage DNA library was screened and a clone expressing a 44 kDa protein was identified. The DNA insert in this clone contained several genes homologous to genes contained in the str operon of Escherichia coli. One ORF product with a calculated molecular mass of 43 kDa was highly homologous to the EF-TuA of E. coli. A synthetic peptide antiserum specific for a portion of the C terminus of EF-Tu confirmed that the 37 kDa protein in whole-cell lysates of N. gonorrhoeae was a processed form of EF-Tu. Deletion of the tufA gene homologue in N. gonorrhoeae was attempted but was unsuccessful. PMID:8828215

  17. Effect of radiation processing on in vitro protein digestibility and availability of calcium, phosphorus and iron of peanut

    Science.gov (United States)

    Hassan, Amro B.; Diab, Eiman E.; Mahmoud, Nagat S.; Elagib, Randa A. A.; Rushdi, Mohamed A. H.; Osman, Gammaa A. M.

    2013-10-01

    The effect of gamma irradiation of two peanut cultivars (Sodari and Madani) on protein content, in vitro protein digestibility and availability of calcium, phosphorus and iron was determined. Seeds were treated with gamma irradiation at dose levels of 1.0, 1.5 and 2.0 kGy. Total protein in seeds was not changed significantly by irradiation. However, the in vitro protein digestibility was decreased for both cultivars. In addition, the irradiation also caused an increment on the available calcium, phosphorus and iron for both cultivars. Moreover, radiation processing caused an increment on tannin content of the seeds especially at the dose 2 kGy for both cultivars. Regarding these results, irradiation treatment of peanut up to 2 kGy can be used as an effective alternative method to chemical treatments for insect disinfestation and microbial disinfection.

  18. IMAGING OF FLUOROPHORES IN CHROMATOGRAPHIC BEADS, RECONSTRUCTION OF RADIAL DENSITY DISTRIBUTIONS AND CHARACTERISATION OF PROTEIN UPTAKING PROCESSES

    Directory of Open Access Journals (Sweden)

    Bernd Stanislawski

    2010-11-01

    Full Text Available A new adjustment calculus is presented to determine the true intraparticle distribution of bound protein within chromatographic beads from confocal fluorescence slice series. The calculus does not require knowledge about optical properties of different chromatographic materials like refractive index and turbidity, but it depends on a parameter which can be adjusted interactively. The algorithm is of complexity O(n where n is the pixel number. From the reconstructed data we compute the parameters of the protein uptaking process using a model-based approach. It is demonstrated that the protein uptaking rates of the beads strongly dependent on the conditions of the fluid phase influencing the strength of protein surface interaction.

  19. Study Technological Factors Effect on the Loss of Protein, Carbohydrate and Lipid inside Royal Jelly in the Freeze Drying Process

    OpenAIRE

    Nguyen Tan Dzung; Le Duc Manh; Nguyen Van Suc

    2015-01-01

    This study published the mathematical models that were built by experiment to describe relationships between the loss of nutritional substances such as protein, carbohydrate and lipid of Royal jelly with technological factors in the freeze drying process such as temperature and pressure of freeze drying chamber; time of freeze drying process. These relationships were applied to determine the optimal technological factors. The results were found out the optimal technological mode as follow: th...

  20. The effect of the application of protein and cellulose preparations as iodine carriers on stability of thiamine in processed meats

    OpenAIRE

    Krystyna Szymandera-Buszka; Katarzyna Waszkowiak; Marzanna Hęś; Anna Jędrusek-Golińska

    2011-01-01

      Fortification of processed meat with iodised table salt was shown to increase thiamine losses, both during thermal processing and storage. Taking into consideration the fact, as well as the recommendation for reduction of consumption of table salt, alternative iodine carriers need to be searched for. Thus the aim of the study was to determine the effect of soy protein isolate (SPI) and wheat fibre (WF) as iodine salts’ (potassium iodide and iodate) carriers on thiamine stabil...

  1. Processed vs. Non-Processed Biowastes for Agriculture: Effects of Post-Harvest Tomato Plants and Biochar on Radish Growth, Chlorophyll Content and Protein Production

    Science.gov (United States)

    Mozzetti Monterumici, Chiara; Rosso, Daniele; Montoneri, Enzo; Ginepro, Marco; Baglieri, Andrea; Novotny, Etelvino Henrique; Kwapinski, Witold; Negre, Michèle

    2015-01-01

    The aim of this work was to address the issue of processed vs. non-processed biowastes for agriculture, by comparing materials widely differing for the amount of process energy consumption. Thus, residual post harvest tomato plants (TP), the TP hydrolysates obtained at pH 13 and 60 °C, and two known biochar products obtained by 650 °C pyrolysis were prepared. All products were characterized and used in a cultivation of radish plants. The chemical composition and molecular nature of the materials was investigated by solid state 13C NMR spectrometry, elemental analysis and potentiometric titration. The plants were analysed for growth and content of chlorophyll, carotenoids and soluble proteins. The results show that the TP and the alkaline hydrolysates contain lignin, hemicellulose, protein, peptide and/or amino acids moieties, and several mineral elements. The biochar samples contain also similar mineral elements, but the organic fraction is characterized mainly by fused aromatic rings. All materials had a positive effect on radish growth, mainly on the diameter of roots. The best performances in terms of plant growth were given by miscanthus originated biochar and TP. The most significant effect was the enhancement of soluble protein content in the plants treated with the lowest energy consumption non processed TP. The significance of these findings for agriculture and the environment is discussed. PMID:25906472

  2. Allerginicity of wheat proteins: the effect of thermal processing (baking and cooking), high pressure treatment and enzymatic digestion

    Czech Academy of Sciences Publication Activity Database

    Kitanovičová, Andrea; Janatková, I.; Houska, M.; Tučková, Ludmila

    Latina : Allergy data laboratories, 2007, s. 43-43. [International Symposium on Molecular Allergology /2./. Roma (IT), 22.04.2007-24.04.2007] R&D Projects: GA ČR GA310/05/2245 Institutional research plan: CEZ:AV0Z50200510 Keywords : food allergy * wheat proteins * thermal processing Subject RIV: EE - Microbiology, Virology

  3. Processing and food chemistry to improve protein ingredients of plant origin for aquafeeds

    Science.gov (United States)

    Developing alternative protein ingredients for aquafeeds that support fish growth and are economically feasible has been a primary task for world aquaculture. Due to abundant production, oilseeds and cereals have been the major sources of alternative proteins. Oilseeds, such as soybeans, have high...

  4. Starch extraction process coupled to protein recovery from leguminous tuberous roots (Pachyrhizus ahipa).

    Science.gov (United States)

    Díaz, Andrea; Dini, Cecilia; Viña, Sonia Z; García, María A

    2016-11-01

    The objective of this work was to fit together the starch extraction from Pachyrhizus ahipa roots and the recovery of the proteins present in these storage organs, making an improved use of this novel raw material. The replacement of water by buffer PO4(-3)/NaCl as solvent in the first extraction steps improved protein extraction without lowering the starch yield. The starches obtained from the traditional and the proposed methods exhibited some differences in appearance and technological and thermal properties, which were endorsed to the adjustment in the methodology of extraction rather than to the use of buffer as solvent. Thus, P. ahipa starch obtaining procedure could be coupled to protein extraction with a minimum change in the methodology. This innovation did not significantly shift the characteristics of the starch obtained and allowed to obtain a protein yield of 135.7mg BSA equivalent protein/100g of fresh roots. PMID:27516269

  5. Influence of sodium nitrite on protein oxidation and nitrosation of sausages subjected to processing and storage.

    Science.gov (United States)

    Feng, Xianchao; Li, Chenyi; Jia, Xu; Guo, Yan; Lei, Na; Hackman, Robert M; Chen, Lin; Zhou, Guanghong

    2016-06-01

    The influence of NaNO2 content on protein oxidation and nitrosation was investigated in cooked sausages at different concentrations (0, 50, 100, 200 and 400mg NaNO2/kg). Dependent on concentration, NaNO2 had both anti- and pro-oxidant effects on protein oxidation. The antioxidant effects of NaNO2 on the protein oxidation were evidenced by significantly lower carbonyl contents, higher free amines and lower surface hydrophobicities. The pro-oxidant effects of NaNO2 on protein oxidation resulted in a decrease of sulfhydryls and an increase of disulfide bonds. NaNO2 also improved the protein nitrosation inducing the formation of 3-nitrotyrosine (3-NT). Moreover, 3-NT had significant correlations with parameters of protein oxidation, indicating that 3-NT may be a possible marker for protein oxidation. Results of this study contribute to an understanding of the impact of NaNO2 on food quality and help to identify optimal formulations of cured meat products. PMID:26923991

  6. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

    International Nuclear Information System (INIS)

    Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNVKUN) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNVKUN particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNVKUN under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNVKUN assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNVKUN assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system. - Highlights: • We show that the ISG MxA can recognize the West Nile virus capsid protein. • Interaction between WNV C protein and MxA induces cytoplasmic fibrils. • MxA can be retargeted to the ER to restrict WNV particle release. • WNV assembly process is a strategy to avoid MxA recognition

  7. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

    Energy Technology Data Exchange (ETDEWEB)

    Hoenen, Antje [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane (Australia); Gillespie, Leah [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia); Morgan, Garry; Heide, Peter van der [Institute for Molecular Bioscience, University of Queensland, Brisbane (Australia); Khromykh, Alexander [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane (Australia); Australian Infectious Diseases Research Centre, University of Queensland, Brisbane (Australia); Mackenzie, Jason, E-mail: jason.mackenzie@unimelb.edu.au [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia)

    2014-01-05

    Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNV{sub KUN}) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNV{sub KUN} particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNV{sub KUN} under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNV{sub KUN} assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNV{sub KUN} assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system. - Highlights: • We show that the ISG MxA can recognize the West Nile virus capsid protein. • Interaction between WNV C protein and MxA induces cytoplasmic fibrils. • MxA can be retargeted to the ER to restrict WNV particle release. • WNV assembly process is a strategy to avoid MxA recognition.

  8. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    Science.gov (United States)

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  9. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus.

    OpenAIRE

    Stark, R; Meyers, G; Rümenapf, T.; Thiel, H J

    1993-01-01

    The polyprotein of classical swine fever virus starts with the nonstructural protein p23, which is followed by the nucleocapsid protein p14. Proteolytic cleavage between p23 and p14 was demonstrated in a cell-free transcription-translation system. Successive truncation of the cDNA used for the transcription indicated that the proteolytic activity responsible for the cleavage between p23 and p14 resides within p23. In order to determine the cleavage site between these two proteins, the respect...

  10. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

    Science.gov (United States)

    Park, Richard; El-Guindy, Ayman; Heston, Lee; Lin, Su-Fang; Yu, Kuan-Ping; Nagy, Mate; Borah, Sumit; Delecluse, Henri-Jacques; Steitz, Joan; Miller, George

    2014-01-01

    Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors. PMID:24705134

  11. Control tools to detect processed animal proteins in feed and in animal by-products: specificity and challenges

    Directory of Open Access Journals (Sweden)

    Woodgate SL.

    2009-01-01

    Full Text Available AbstractThis paper reviews the current situation with regard to a total feed ban on the use of processed animal proteins in feed for meat producing animals within the EU. The scientific aspects surrounding the development of control tools are discussed. In particular, focus is given to methods for marking those materials prohibited in animal feeds and for the determination of species specificity in those proteins that are potentially allowed in animal feeds. The overall objective is that the advancements in science are utilized to achieve a partial relaxation of the total feed ban in the near future.

  12. Study of quantitative changes of cereal allergenic proteins after food processing

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Benkovská, Dagmar; Laštovičková, Markéta

    2015-01-01

    Roč. 95, č. 5 (2015), s. 983-990. ISSN 0022-5142 Institutional support: RVO:68081715 Keywords : allergens * proteins * barley * mass spectrometry Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.714, year: 2014

  13. Effect of Silk Protein Processing on Drug Delivery from Silk Films

    OpenAIRE

    Pritchard, Eleanor M.; Hu, Xiao; Finley, Violet; Kuo, Catherine K.; Kaplan, David L.

    2013-01-01

    Sericin removal from the core fibroin protein of silkworm silk is a critical first step in the use of silk for biomaterial-related applications, but degumming can affect silk biomaterial properties, including molecular weight, viscosity, diffusivity and degradation behavior. Increasing the degumming time (10, 30, 60 and 90 min) decreases the average molecular weight of silk protein in solution, silk solution viscosity, and silk film glass transition temperature, and increases the rate of degr...

  14. Subacute Changes in Cleavage Processing of Amyloid Precursor Protein and Tau following Penetrating Traumatic Brain Injury

    Science.gov (United States)

    Mountney, Andrea; Hwang, Hye; Swiercz, Adam; Rammelkamp, Zoe; Boutte, Angela M.; Shear, Deborah A.; Tortella, Frank C.; Schmid, Kara E.

    2016-01-01

    Traumatic brain injury (TBI) is an established risk factor for the development of Alzheimer’s disease (AD). Here the effects of severe penetrating TBI on APP and tau cleavage processing were investigated in a rodent model of penetrating ballistic-like brain injury (PBBI). PBBI was induced by stereotactically inserting a perforated steel probe through the right frontal cortex of the anesthetized rat and rapidly inflating/deflating the probe’s elastic tubing into an elliptical shaped balloon to 10% of total rat brain volume causing temporary cavitation injury. Separate animals underwent probe injury (PrI) alone without balloon inflation. Shams underwent craniectomy. Brain tissue was collected acutely (4h, 24h, 3d) and subacutely (7d) post-injury and analyzed by immunoblot for full length APP (APP-FL) and APP beta c-terminal fragments (βCTFs), full length tau (tau-FL) and tau truncation fragments and at 7d for cytotoxic Beta amyloid (Aβ) peptides Aβ40 and Aβ42 analysis. APP-FL was significantly decreased at 3d and 7d following PBBI whereas APP βCTFs were significantly elevated by 4h post-injury and remained elevated through 7d post-injury. Effects on βCTFs were mirrored with PrI, albeit to a lesser extent. Aβ40 and Aβ42 were significantly elevated at 7d following PBBI and PrI. Tau-FL decreased substantially 3d and 7d post-PBBI and PrI. Importantly, a 22 kDa tau fragment (tau22), similar to that found in AD, was significantly elevated by 4h and remained elevated through 7d post-injury. Thus both APP and tau cleavage was dramatically altered in the acute and subacute periods post-injury. As cleavage of these proteins has also been implicated in AD, TBI pathology shown here may set the stage for the later development of AD or other tauopathies. PMID:27428544

  15. Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

    OpenAIRE

    Henry Ryan E; Muto Machiko; Mayfield Stephen P

    2009-01-01

    Abstract Background Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing ...

  16. Identification of Proteins and Peptide Biomarkers for Detecting Banned Processed Animal Proteins (PAPs) in Meat and Bone Meal by Mass Spectrometry.

    Science.gov (United States)

    Marbaix, Hélène; Budinger, Dimitri; Dieu, Marc; Fumière, Olivier; Gillard, Nathalie; Delahaut, Philippe; Mauro, Sergio; Raes, Martine

    2016-03-23

    The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with processed animal proteins (PAPs) as the main vector of the disease, has led to their prohibition in feed. The progressive release of the feed ban required the development of new analytical methods to determine the exact origin of PAPs from meat and bone meal. We set up a promising MS-based method to determine the species and the source (legal or not) present in PAPs: a TCA-acetone protein extraction followed by a cleanup step, an in-solution tryptic digestion of 5 h (with a 1:20 protein/trypsin ratio), and mass spectrometry analyses, first without any a priori, with a Q-TOF, followed by a targeted triple-quadrupole analysis. Using this procedure, we were able to overcome some of the major limitations of the official methods to analyze PAPs, detecting and identifying prohibited animal products in feedstuffs by the monitoring of peptides specific for cows, pigs, and sheep in PAPs. PMID:26943838

  17. The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

    International Nuclear Information System (INIS)

    A large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses

  18. Respiratory Syncytial Virus (RSV) Infects Neuronal Cells and Processes That Innervate the Lung by a Process Involving RSV G Protein

    OpenAIRE

    Li, Xia-qing; Fu, Zhen F.; Alvarez, Rene; Henderson, Christine; Ralph A. Tripp

    2006-01-01

    Respiratory syncytial virus (RSV) is a primary cause of morbidity and life-threatening lower respiratory tract disease in infants and young children. Children with acute RSV bronchiolitis often develop respiratory sequelae, but the disease mechanisms are poorly understood. Mounting evidence suggests that RSV may mediate persistent infection. Using immunohistochemistry to identify RSV and RSV-infected cell types, we show that RSV infects primary neurons and neuronal processes that innervate th...

  19. The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins

    Science.gov (United States)

    Rouillard, Andrew D.; Gundersen, Gregory W.; Fernandez, Nicolas F.; Wang, Zichen; Monteiro, Caroline D.; McDermott, Michael G.; Ma’ayan, Avi

    2016-01-01

    Genomics, epigenomics, transcriptomics, proteomics and metabolomics efforts rapidly generate a plethora of data on the activity and levels of biomolecules within mammalian cells. At the same time, curation projects that organize knowledge from the biomedical literature into online databases are expanding. Hence, there is a wealth of information about genes, proteins and their associations, with an urgent need for data integration to achieve better knowledge extraction and data reuse. For this purpose, we developed the Harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins from over 70 major online resources. We extracted, abstracted and organized data into ∼72 million functional associations between genes/proteins and their attributes. Such attributes could be physical relationships with other biomolecules, expression in cell lines and tissues, genetic associations with knockout mouse or human phenotypes, or changes in expression after drug treatment. We stored these associations in a relational database along with rich metadata for the genes/proteins, their attributes and the original resources. The freely available Harmonizome web portal provides a graphical user interface, a web service and a mobile app for querying, browsing and downloading all of the collected data. To demonstrate the utility of the Harmonizome, we computed and visualized gene–gene and attribute–attribute similarity networks, and through unsupervised clustering, identified many unexpected relationships by combining pairs of datasets such as the association between kinase perturbations and disease signatures. We also applied supervised machine learning methods to predict novel substrates for kinases, endogenous ligands for G-protein coupled receptors, mouse phenotypes for knockout genes, and classified unannotated transmembrane proteins for likelihood of being ion channels. The Harmonizome is a comprehensive resource of knowledge

  20. The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins.

    Science.gov (United States)

    Rouillard, Andrew D; Gundersen, Gregory W; Fernandez, Nicolas F; Wang, Zichen; Monteiro, Caroline D; McDermott, Michael G; Ma'ayan, Avi

    2016-01-01

    Genomics, epigenomics, transcriptomics, proteomics and metabolomics efforts rapidly generate a plethora of data on the activity and levels of biomolecules within mammalian cells. At the same time, curation projects that organize knowledge from the biomedical literature into online databases are expanding. Hence, there is a wealth of information about genes, proteins and their associations, with an urgent need for data integration to achieve better knowledge extraction and data reuse. For this purpose, we developed the Harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins from over 70 major online resources. We extracted, abstracted and organized data into ∼72 million functional associations between genes/proteins and their attributes. Such attributes could be physical relationships with other biomolecules, expression in cell lines and tissues, genetic associations with knockout mouse or human phenotypes, or changes in expression after drug treatment. We stored these associations in a relational database along with rich metadata for the genes/proteins, their attributes and the original resources. The freely available Harmonizome web portal provides a graphical user interface, a web service and a mobile app for querying, browsing and downloading all of the collected data. To demonstrate the utility of the Harmonizome, we computed and visualized gene-gene and attribute-attribute similarity networks, and through unsupervised clustering, identified many unexpected relationships by combining pairs of datasets such as the association between kinase perturbations and disease signatures. We also applied supervised machine learning methods to predict novel substrates for kinases, endogenous ligands for G-protein coupled receptors, mouse phenotypes for knockout genes, and classified unannotated transmembrane proteins for likelihood of being ion channels. The Harmonizome is a comprehensive resource of knowledge about

  1. Process strategies to improve heterologous protein production in Escherichia coli under lactose or IPTG induction

    DEFF Research Database (Denmark)

    Kilikian, B. V.; Surarez, I. D.; Liria, C. W.; Gombert, Andreas Karoly

    2000-01-01

    Cells of Escherichia coli BL21 bearing the chicken muscle Troponin C (TnC) gene under the control of the lacUV5 promoter were induced under different cultivation conditions and the consequences on growth and cell protein content were investigated. The type of inducer molecule (lactose or IPTG), the...... per gram dry cell weight (DCW), was achieved when isopropyl-beta-D-thiogalactoside (IPTG) was the inducer. Under lactose induction, a value of 96 mg per gram DCW was attained. However, the high metabolic load imposed by IPTG, when compared with lactose induction, as assessed by the cell protein...... content and stability, indicates that lactose is probably the most appropriate inducer for the synthesis of this heterologous protein. (C) 2000 Elsevier Science Ltd. All rights reserved....

  2. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

    DEFF Research Database (Denmark)

    de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, F;

    2014-01-01

    plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium......-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL-1 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration...... in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher...

  3. Polyunsaturated Fatty Acids Selectively Suppress Sterol Regulatory Element-binding Protein-1 through Proteolytic Processing and Autoloop Regulatory Circuit*

    OpenAIRE

    Takeuchi, Yoshinori; Yahagi, Naoya; Izumida, Yoshihiko; Nishi, Makiko; Kubota, Midori; Teraoka, Yuji; Yamamoto, Takashi; Matsuzaka, Takashi; Nakagawa, Yoshimi; Sekiya, Motohiro; Iizuka, Yoko; Ohashi, Ken; Osuga, Jun-ichi; Gotoda, Takanari; Ishibashi, Shun

    2010-01-01

    Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for P...

  4. Protein crystallization as a process step in a novel meso oscillatory flow reactor: study of lysozyme phase behavior

    OpenAIRE

    Castro, Filipa; Ferreira, António; Teixeira, J. A.; Rocha, Fernando

    2016-01-01

    In the present work, it is reported for the first time the study of the applicability of a novel meso oscillatory flow reactor (meso-OFR) for protein crystallization as a process step. Crystallization assays carried out in the designed device enabled to derive a two-dimensional lysozyme phase diagram (lysozyme concentration against sodium chloride concentration). Results evidence the formation of several types of crystals (different size and shape), with a strong influence of salt concentrati...

  5. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    NARCIS (Netherlands)

    Maatz, H.; Jens, M.; Liss, M.; Schafer, S.; Heinig, M.; Kirchner, M.; Adami, E.; Rintisch, C.; Dauksaite, V.; Radke, M.H.; Selbach, M.; Barton, P.J.; Cook, S.A.; Rajewsky, N.; Gotthardt, M.; Landthaler, M.; Hubner, N.

    2014-01-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and

  6. Industry perspective on the validation of column-based separation processes for the purification of proteins. Parenteral Drug Association.

    Science.gov (United States)

    1992-01-01

    Validation of column-based separations is necessary to ensure the quality and safety of protein and peptide products produced by rDNA, peptide synthesis, and hybridoma technologies. Process validation for column-based separations includes qualification of raw materials, equipment, and the purification process. Combined with in-process control and quality control of the final product, column validation ensures that a uniform product is produced consistently from batch to batch. In the best case, validation is designed into the process. During process design, techniques are selected which can remove impurities and contaminants. Equipment and chromatographic media which can perform reproducibly are selected. Column performance standards, cleaning and regeneration routines, and column life should be considered as early as possible. Clearance studies should be planned and implemented to ensure that a product is produced with the requisite purity. There are no explicit rules for process validation of column-based separation processes. This document is intended to serve as a starting point for those needing to validate column-based separation processes. PMID:1522447

  7. Poly(A)-binding proteins are required for diverse biological processes in metazoans.

    Science.gov (United States)

    Smith, Richard W P; Blee, Tajekesa K P; Gray, Nicola K

    2014-08-01

    PABPs [poly(A)-binding proteins] bind to the poly(A) tail of eukaryotic mRNAs and are conserved in species ranging from yeast to human. The prototypical cytoplasmic member, PABP1, is a multifunctional RNA-binding protein with roles in global and mRNA-specific translation and stability, consistent with a function as a central regulator of mRNA fate in the cytoplasm. More limited insight into the molecular functions of other family members is available. However, the consequences of disrupting PABP function in whole organisms is less clear, particularly in vertebrates, and even more so in mammals. In the present review, we discuss current and emerging knowledge with respect to the functions of PABP family members in whole animal studies which, although incomplete, already underlines their biological importance and highlights the need for further intensive research in this area. PMID:25110030

  8. UVA-induced damage to DNA and proteins: direct versus indirect photochemical processes

    Science.gov (United States)

    Girard, P. M.; Francesconi, S.; Pozzebon, M.; Graindorge, D.; Rochette, P.; Drouin, R.; Sage, E.

    2011-01-01

    UVA has long been known for generating an oxidative stress in cells. In this paper we review the different types of DNA damage induced by UVA, i.e. strand breaks, bipyrimidine photoproducts, and oxidatively damaged bases. Emphasis is given to the mechanism of formation that is further illustrated by the presentation of new in vitro data. Examples of oxidation of proteins involved in DNA metabolism are also given.

  9. UVA-induced damage to DNA and proteins: direct versus indirect photochemical processes

    International Nuclear Information System (INIS)

    UVA has long been known for generating an oxidative stress in cells. In this paper we review the different types of DNA damage induced by UVA, i.e. strand breaks, bipyrimidine photoproducts, and oxidatively damaged bases. Emphasis is given to the mechanism of formation that is further illustrated by the presentation of new in vitro data. Examples of oxidation of proteins involved in DNA metabolism are also given.

  10. Aqueous two phase extraction of proteins: From molecular understanding to process development

    OpenAIRE

    Oelmeier, Stefan A

    2012-01-01

    A high throughput screening method was implemented and applied to an industrial separation task. Previously reported correlations between protein descriptors and distribution were evaluated. A new approach to screen ATPS for their industrial application was devised and put to use for the selection of ATPSs used in centrifugal partitioning chromatography. A new modeling approach based on molecular dynamics was set up. This approach was validated using single PEG molecules in solution.

  11. Protein kinase C interaction with calcium: a phospholipid-dependent process.

    LENUS (Irish Health Repository)

    Bazzi, M D

    1990-08-21

    The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

  12. Ligation of FcγR Alters Phagosomal Processing of Protein via Augmentation of NADPH Oxidase Activity.

    Science.gov (United States)

    Balce, Dale R; Rybicka, Joanna M; Greene, Catherine J; Ewanchuk, Benjamin W; Yates, Robin M

    2016-07-01

    Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population-based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G (IgG)-stimulated reactive oxygen species (ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C (PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR-mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG-opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR-triggered, PKC/Syk-dependent local assembly and activation of NOX2. PMID:27020146

  13. The beta-propeller protein YxaL increases the processivity of the PcrA helicase.

    Science.gov (United States)

    Noirot-Gros, M-F; Soultanas, P; Wigley, D B; Ehrlich, S D; Noirot, P; Petit, M-A

    2002-05-01

    The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component. PMID:12073041

  14. Study of factors that interfere in the labelling process of erythrocytes and plasma proteins with Technetium-99m

    International Nuclear Information System (INIS)

    The labelling of red blood cells (RBC) with technetium-99m (Tc-99m) depends on several factors, as the stannous ion (Sn++) concentration, time, temperature, the presence of plasma proteins (PP) and others. However the Sn++ concentration seems to be the most important factor; probably because the uptake of this reducing agent by RBC is limited. The excess of Sn++ in extracellular medium can determine the labelling of PP. the modifications of RBC at 50 deg C described in the literature, the possibility of labelling RBC with Tc-99m at this temperature and experimental results obtained made it possible to perform spleen selective scintigraphy through a simple technique with few manipulations. The effect of gentamicin, nifedipine and verapamil in the labelling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca++ and Sn++. The results show that, under some conditions, these drugs are capable to alter this Tc-99m incorporation. The modification of the ionic distribution determined by these drugs or the blockage of Sn++ and/or Tc-99m or the fact that they bind theirselves to plasma proteins, or the possibility of the labelling of these drugs, are factors that can interfere in the labelling process of red blood cells and plasma proteins with Tc-99m. (author)

  15. Assessment of the sensitizing potential of processed peanut proteins in Brown Norway rats: roasting does not enhance allergenicity.

    Directory of Open Access Journals (Sweden)

    Stine Kroghsbo

    Full Text Available BACKGROUND: IgE-binding of process-modified foods or proteins is the most common method for examination of how food processing affects allergenicity of food allergens. How processing affects sensitization capacity is generally studied by administration of purified food proteins or food extracts and not allergens present in their natural food matrix. OBJECTIVES: The aim was to investigate if thermal processing increases sensitization potential of whole peanuts via the oral route. In parallel, the effect of heating on sensitization potential of the major peanut allergen Ara h 1 was assessed via the intraperitoneal route. METHODS: Sensitization potential of processed peanut products and Ara h 1 was examined in Brown Norway (BN rats by oral administration of blanched or oil-roasted peanuts or peanut butter or by intraperitoneal immunization of purified native (N-, heated (H- or heat glycated (G-Ara h 1. Levels of specific IgG and IgE were determined by ELISA and IgE functionality was examined by rat basophilic leukemia (RBL cell assay. RESULTS: In rats dosed orally, roasted peanuts induced significant higher levels of specific IgE to NAra h 1 and 2 than blanched peanuts or peanut butter but with the lowest level of RBL degranulation. However, extract from roasted peanuts was found to be a superior elicitor of RBL degranulation. Process-modified Ara h 1 had similar sensitizing capacity as NAra h 1 but specific IgE reacted more readily with process-modified Ara h 1 than with native. CONCLUSIONS: Peanut products induce functional specific IgE when dosed orally to BN rats. Roasted peanuts do not have a higher sensitizing capacity than blanched peanuts. In spite of this, extract from roasted peanuts is a superior elicitor of RBL cell degranulation irrespectively of the peanut product used for sensitization. The results also suggest that new epitopes are formed or disclosed by heating Ara h 1 without glucose.

  16. Effect of an Antimicrobial Compound on Different Processes within the Oscillation of Min Proteins in E. coli Bacterial Cells

    Science.gov (United States)

    Giuliani, Maximiliano; Dutcher, John

    2013-03-01

    A key step in the life of a bacterium is its division into two daughter cells of equal size. This process is carefully controlled and regulated so that equal partitioning of the cellular machinery is obtained. In E. coli, this regulation is accomplished, in part, by the Min protein system. The Min proteins undergo an oscillation between the poles of rod-shaped E. coli bacteria. We use high magnification, time-resolved total internal reflection fluorescence microscopy to characterize the temporal distributions of different processes within the oscillation: the MinD-MinE interaction time, the residence time for membrane bound MinD, and the recruitment time for MinD to be observed at the opposite pole. We also characterize the change in each of these processes in the presence of the antimicrobial compound polymyxin B (PMB). We show that the times corresponding to the removal of MinD from one pole and the recruitment of MinD at the opposite pole are correlated. We explain this correlation through the existence of a concentration threshold. The effect of PMB on the concentration threshold is used to identify which process within the oscillation is most affected.

  17. Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin.

    Science.gov (United States)

    Nuchuchua, O; Every, H A; Jiskoot, W

    2016-06-01

    The aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO2 spray drying processing parameters. Myoglobin was used as a model protein (5mg/ml with 50mg/ml trehalose in 10mM phosphate buffer, pH 6.2). The solution was exposed to sub- and supercritical CO2 conditions (65-130bar and 25-50°C), and CO2 spray drying under those conditions. The heme binding of myoglobin was determined by UV/Vis, fluorescence, and circular dichroism spectroscopy, while myoglobin aggregation was studied by using size-exclusion chromatography and flow imaging microscopy. It was found that pressure and temperature alone did not influence myoglobin's integrity. However, when pressurized CO2 was introduced into myoglobin solutions at any condition, the pH of the myoglobin formulation shifted to about 5 (measured after depressurization), resulting in heme binding destabilization and aggregation of myoglobin. When exposed to CO2, these degradation processes were enhanced by increasing temperature. Heme binding destabilization and myoglobin aggregation were also seen after CO2 spray drying, and to a greater extent. Moreover, the CO2 spray drying induced the partial loss of heme. In conclusion, pressurized CO2 destabilizes the myoglobin, leading to heme loss and protein aggregation upon spray drying. PMID:27080205

  18. The Human Nucleolar Protein FTSJ3 Associates with NIP7 and Functions in Pre-rRNA Processing

    Science.gov (United States)

    Morello, Luis G.; Coltri, Patricia P.; Quaresma, Alexandre J. C.; Simabuco, Fernando M.; Silva, Tereza C. L.; Singh, Guramrit; Nickerson, Jeffrey A.; Oliveira, Carla C.; Moore, Melissa J.; Zanchin, Nilson I. T.

    2011-01-01

    NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A′ to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells. PMID:22195017

  19. Mammalian cell cultures. Part II: Genetic engineering, protein glycosylation, fermentation and process control.

    Science.gov (United States)

    Werner, R G; Noé, W

    1993-11-01

    For expression of human genes in mammalian cell culture regulatory sequences such as promotor or terminator region of viral origin are required. The most widely used expression system uses dihydrofolic acid reductase (DHFR) as a selection marker in conjunction with a DHFR deficient Chinese hamster ovary (CHO) cell using methotrexate as selection pressure. Alternatively the glutamine synthetase amplification system seems to be one of the most efficient expression systems using methionine sulphoximine (MSX) as selection pressure. Folding and glycosylation takes place in mammalian cell cultures at the sites of endoplasmatic reticulum and the Golgi apparatus and is comparable to synthesis in human cells. Most large scale manufacturing processes for products derived from mammalian cell cultures are fed batch suspension culture processes up to 15,000 l. Important factors for productivity are media composition and feeding strategies. Sterility of the entire system during the fermentation period is a decisive factor for success rate. Because mammalian cell cultures reacting very sensitive to small changes in temperature, pH, osmolality and agitation the fermentation system requires a reliable process control system. Validation of the entire manufacturing process is required to ensure consistent product quality which meets predetermined specifications and to provide a basis for an economic process. In a joint effort equipment qualification, process validation, in-process controls and quality controls provide the basis for product consistency from batch to batch. PMID:8292072

  20. Pulsed laser deposition of the lysozyme protein: an unexpected “Inverse MAPLE” process

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin;

    2012-01-01

    material directly for film production, as in PLD (pulsed laser deposition), where the film molecules may undergo strong fragmentation. In this presentation we report an alternative surprising mechanism for film deposition of the protein lysozyme in vacuum, when a small amount of residual water drives the...... materials. Also, the thermal properties of lysozyme, including the heat-induced decomposition behavior are comparatively well-known. The ablation of lysozyme from a dry pressed target in vacuum was measured by weight loss in nanosecond laser ablation at 355 with a fluence of 0.5 to 6 J/cm2. Films with a...

  1. Influence of the water molecules near surface of viral protein on virus activation process

    International Nuclear Information System (INIS)

    The infection of a cell with influenza virus comprises the stages of receptor binding to the cell membrane, endocytosis of virus particle, and fusion of the virus envelope and cell endosome membrane, which is determined by the conformational changes in hemagglutinin, a virus envelope protein, caused by pH decrease within the endosome. The pH value that induces conformation rearrangements of hemagglutinin molecule considerably varies for different influenza virus strains, first and foremost, due to the differences in amino acid structure of the corresponding proteins. The main goal of this study was to construct a model making it possible to assess the critical pH value characterizing the fusogenic activity of influenza virus hemagglutinin from the data on hemagglutinin structure and experimental verification of this model. Under this model, we assume that when the electrostatic force between interacting hemagglutinin molecules in the virus envelop exceeds a certain value, the hemagglutinin HA1 subunits are arranged so that they form a cavity sufficient for penetration of water molecules. This event leads to an irreversible hydration of the inner fragments of hemagglutinin molecule in a trimer and to the completion of conformational changes. The geometry of electrostatic field in hemagglutinin trimer was calculated taking into account the polarization effects near the interface of two dielectrics, aqueous medium and protein macromolecule. The critical pH values for the conformational changes in hemagglutinin were measured by the erythrocyte hemolysis induced by influenza virus particles when decreasing pH. The critical pH value conditionally separating the pH range into the regions with and without the conformational changes was calculated for several influenza virus H1N1 and H3N2 strains based on the data on the amino acid structure of the corresponding hemagglutinin molecules. Comparison of the theoretical and experimental values of critical pH values for

  2. Seaweed proteins - how to get to them? Effects of processing on nutritional value, bioaccessibility and extractability

    OpenAIRE

    Mæhre, Hanne K

    2016-01-01

    Paper II of this thesis is as published version not available in Munin, but available in Journal of Applied Phycology, April 2015. The accepted manuscript version of paper II is available in Munin: Heat treatment improves the protein bioaccessibility in the red seaweed dulse (Palmaria palmata), but not in the brown seaweed winged kelp (Alaria esculenta). Mæhre HK, Edvinsen GK, Eilertsen K-E & Elvevoll EO.Paper III of this thesis is not available in Munin: Enzymatic pre-treatment increases th...

  3. Biosynthesis of intestinal microvillar proteins. Low temperature arrests both processing and intracellular transport

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge; Cowell, G M

    1989-01-01

    mannose-glycosylated form characteristic of the newly synthesized enzymes prior to the molecular processing taking place in the Golgi complex. The general morphology of the enterocyte was unaffected by culture at low temperature except for the Golgi complex where the cisternae appeared condensed and...... surrounded by numerous vesicles of 50 to 55 nm. Both molecular processing and microvillar expression could be restored by shifting the temperature to 37 degrees C. Culture at low temperature did not induce any missorting of newly synthesized aminopeptidase N, but both molecular processing and microvillar...

  4. Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub-Adult Cichlid Fish, Oreochromis Mossambicus, Reveal Insights Into the Biomineralization Process.

    Science.gov (United States)

    Weigele, Jochen; Franz-Odendaal, Tamara A; Hilbig, Reinhard

    2016-02-01

    The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required. PMID:26559654

  5. Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco.

    Science.gov (United States)

    Buyel, Johannes F; Fischer, Rainer

    2014-02-01

    Flocculation is a cost-effective method that is used to improve the efficiency of clarification by causing dispersed particles to clump together, allowing their removal by sedimentation, centrifugation or filtration. The efficacy of flocculation for any given process depends on the nature and concentration of the particulates in the feed stream, the concentration, charge density and length of the flocculant polymer, the shear rate, the properties of the feed stream (e.g. pH and ionic strength) and the properties of the target products. We tested a range of flocculants and process conditions using a design of experiments approach to identify the most suitable polymers for the clarification step during the production of a HIV-neutralizing monoclonal antibody (2G12) and a fluorescent marker protein (DsRed) expressed in transgenic tobacco leaves. Among the 23 different flocculants we tested, the greatest reduction in turbidity was achieved with Polymin P, a branched, cationic polyethylenimine with a charge density of 13.0 meq/g. This flocculant reduced turbidity by more than 90% under a wide range of process conditions. We developed a model that predicted its performance under different process conditions, and this enabled us to increase the depth filter capacity three-sevenfold depending on the process scale, depth filter type and plant species. The costs of filter consumables were reduced by more than 50% compared with a process without flocculant, and there was no loss of recovery for either 2G12 or DsRed. PMID:24165151

  6. Evaluation of protein undernourishment on the condylar process of the Wistar rat mandible correlation with insulin receptor expression

    Directory of Open Access Journals (Sweden)

    Marcelo Arthur CAVALLI

    2015-04-01

    Full Text Available The mandible condylar process cartilage (CP of Wistar rats is a secondary cartilage and acts as a mandibular growth site. This phenomenon depends on adequate proteins intake and hormone actions, including insulin. Objectives The present study evaluated the morphological aspects and the expression of the insulin receptor (IR in the cartilage of the condylar process (CP of rats subjected to protein undernourishment. Material and Methods The nourished group received a 20% casein diet, while the undernourished group (U received a 5% casein diet. The re-nourished groups, R and RR, were used to assess the effects of re-nutrition during puberty and adulthood, respectively. CPs were processed and stained with picro-sirius red, safranin-O and azocarmine. Scanning electron microscopy and immunohistochemistry were also performed. Results The area of the CP cartilage and the number of cells in the chondroblastic layer decreased in the U group, as did the thickness of the CP layer in the joint and hypertrophic layer. Renourishment during the pubertal stage, but not during the adult phase, restored these parameters. The cell number was restored when re-nutrition occurred in the pubertal stage, but not in the adult phase. The extracellular matrix also decreased in the U group, but was restored by re-nutrition during the pubertal stage and further increased in the adult phase. IR expression was observed in all CPs, being higher in the chondroblastic and hypertrophic cartilage layers. The lowest expression was found in the U and RR groups. Conclusions Protein malnutrition altered the cellularity, the area, and the fibrous cartilage complex, as well as the expression of the IRs.

  7. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  8. Design and processing of nanogels as delivery systems for peptides and proteins

    DEFF Research Database (Denmark)

    Arnfast, Lærke; Madsen, Claus Greve; Jorgensen, Lene;

    2014-01-01

    Nanogels, cross-linked networks of >1 μm in size, are attractive drug-delivery systems, as they not only possess the potential advantages of nanoscale formulations, but also the attractive abilities of a hydrogel; high hydrophilicity, high loading capacity and the potential for biocompatibility and...... controlled release. The focus of this review is to provide an overview of the recent developments within the nanogel field, and how the chemical design of the nanogel polymer has been found to influence the properties of the nanogel system. Novel nanogel systems are discussed with respect to their type of...... cross-linkage and their suitability as therapeutic delivery systems, as well as their ability to stabilize the protein/peptide drug....

  9. Bioactive proteins and energy value of okara as a byproduct in hydrothermal processing of soy milk.

    Science.gov (United States)

    Stanojevic, Sladjana P; Barac, Miroljub B; Pesic, Mirjana B; Jankovic, Vanja S; Vucelic-Radovic, Biljana V

    2013-09-25

    The nutritional properties of raw okara obtained as a byproduct from six soybean varieties during hydrothermal cooking (HTC) of soy milk were assessed. The composition and residual activity (rTIA) of trypsin inhibitors (TIs), contents of lectin, proteins, fats, and carbohydrates, and energy values (EV) were correlated with the respective physicochemical properties of soybean and okara. Kunitz (KTI) and Bowman-Birk (BBI) TIs both comprised okara rTIA. TIs content was higher in okara (5.19-14.40%) than in soybean (3.10-12.17%), which additionally enriched okara by cysteine. Contents of KTI (r = 1.00;p antinutritional factors. The proximate composition of raw okara, advantageous rTIA, and a very low EV (2.74-3.78 kJ/g) qualify this byproduct for potential application in food preparation as a functional ingredient in dietary products. PMID:23978042

  10. Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme.

    Science.gov (United States)

    Comstock, Matthew J; Whitley, Kevin D; Jia, Haifeng; Sokoloski, Joshua; Lohman, Timothy M; Ha, Taekjip; Chemla, Yann R

    2015-04-17

    The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed "closed" and "open," correlate with movement toward or away from the DNA fork. PMID:25883359

  11. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  12. LASIK surgery of granular corneal dystrophy type 2 patients leads to accumulation and differential proteolytic processing of transforming growth factor beta-induced protein (TGFBIp)

    DEFF Research Database (Denmark)

    Poulsen, Ebbe Toftgaard; Nielsen, Nadia Sukusu; Jensen, Morten M;

    2016-01-01

    laser-assisted in situ keratomileusis (LASIK) surgery protein accumulation is accelerated and they develop massive protein accumulations a few years after surgery. Here, we present the protein profile of aggregate-containing corneal tissue from GCD2 patients with a history of LASIK surgery using LC...... LASIK surgery, which may be important for the disease progression. Lastly, 2DE also revealed differential processing between GCD2 patients with a history of LASIK surgery when compared to healthy individuals....

  13. Model-based high-throughout process development for chromatographic whey proteins separation

    NARCIS (Netherlands)

    Nfor, B.; Ripic, J.; Padt, van der A.; Jacobs, M.; Ottens, M.

    2012-01-01

    In this study, an integrated approach involving the combined use of high-throughput screening (HTS) and column modeling during process development was applied to an industrial case involving the evaluation of four anion-exchange chromatography (AEX) resins and four hydrophobic interaction chromatogr

  14. Optimal Design of Algae Biorefinery Processing Networks for the production of Protein, Ethanol and Biodiesel

    DEFF Research Database (Denmark)

    Cheali, Peam; Vivion, Anthony; Gernaey, Krist V.;

    2015-01-01

    analysis such as microalgae production cost, composition of microalgae (e.g. oil content) and biodiesel/bioethanol market prices is considered. New optimal processing paths are found with potential of producing higher amount of biodiesel. Last, the methodology is intended as decision support tool for early...

  15. Blue copper proteins as a model for investigating electron transfer processes within polypeptide matrices

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I

    1994-01-01

    Cu(II) ion bound at a distance of approximately 2.6 nm has been studied, in naturally occurring and in single-site mutated azurins. The role of changing specific amino acid residues on the internal long-range electron transfer (LRET) process and its potential pathways has been investigated. It is...

  16. Autoantibodies to protein transport and messenger RNA processing pathways: endosomes, lysosomes, Golgi complex, proteasomes, assemblyosomes, exosomes, and GW bodies.

    Science.gov (United States)

    Stinton, Laura M; Eystathioy, Theophany; Selak, Sanja; Chan, Edward K L; Fritzler, Marvin J

    2004-01-01

    Over 50 years ago the lupus erythematosus (LE) cell phenomenon was described and this was quickly followed by the introduction of the LE cell test and indirect immunofluorescence (IIF) to detect antinuclear antibodies (ANA) in clinical laboratories. Recently, attention has turned to the identification of the autoantigens that bind to cytoplasmic organelles such as the Golgi complex, endosomes and other "cytoplasmic somes". Three endosome autoantigens include early endosome antigen 1 (EEA1, 160 kDa), cytoplasmic linker protein-170 (CLIP-170, 170 kDa), and lysobisphosphatidic acid (LBPA). Antibodies to EEA1 were seen in a variety of conditions but approximately 40% of the patients had a neurological disease. Despite the prominence of lysosomes in cells and tissues, reports of autoantibodies are limited to the lysosomal antigen h-LAMP-2 and the cytoplasmic antineutrophil antibodies (cANCA). Autoantigens in the Golgi complex include giantin/macrogolgin, golgin-245, golgin 160, golgin-97, golgin 95/gm130, and golgin-67. More recently, there has been an interest in autoantibodies that bind components of the "SMN complex" or the "assemblyosome". Arginine/glycine (RG)-rich domains in components of the SMN complex interact with Sm, like-Sm (LSm), fibrillarin, RNA helicase A (Gu), and coilin proteins, all of which are antigen targets in a variety of diseases. More recently, components of a novel cytoplasmic structure named GW bodies (GWBs) have been identified as targets of human autoantibodies. Components of GWBs include GW182, a unique mRNA-binding protein, like Sm proteins (LSms), and decapping (hDcp1) and exonuclease (Xrn) enzymes. Current evidence suggests that GWBs are involved in the cytoplasmic processing of mRNAs. Autoantibodies to the "cytoplasmic somes" are relatively uncommon and serological tests to detect most of them are not widely available. PMID:14962794

  17. Chiral recognition and selection during the self-assembly process of protein-mimic macroanions

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Panchao; Zhang, Zhi-Ming; Lv, Hongjin; Li, Tao; Haso, Fadi; Hu, Lang; Zhang, Baofang; Basca, John; Wei, Yongge; Gao, Yanqing; Hou, Yu; Li, Yang-Guang; Hill, Craig L.; Wang, En-Bo; Liu, Tianbo

    2015-03-01

    The research on chiral recognition and chiral selection is not only fundamental in resolving the puzzle of homochirality, but also instructive in chiral separation and stereoselective catalysis. Here we report the chiral recognition and chiral selection during the self-assembly process of two enantiomeric wheel-shaped macroanions, [Fe28(μ3-O)8(Tart)16(HCOO)24]20- (Tart=D- or L-tartaric acid tetra-anion). The enantiomers are observed to remain self-sorted and self-assemble into their individual assemblies in their racemic mixture solution. The addition of chiral co-anions can selectively suppress the self-assembly process of the enantiomeric macroanions, which is further used to separate the two enantiomers from their mixtures on the basis of the size difference between the monomers and the assemblies. We believe that delicate long-range electrostatic interactions could be responsible for such high-level chiral recognition and selection.

  18. Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

    DEFF Research Database (Denmark)

    Danielsen, E M; Norén, Ove; Sjöström, H

    1983-01-01

    that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in......The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane......-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was...

  19. PROTEIN STRUCTURE: A OBSTACLE TO THE UNDERSTANDING OF BIOCHEMICAL PROCESSES FOR HIGH SCHOOL TEACHERS

    Directory of Open Access Journals (Sweden)

    S. L. Menezes

    2007-05-01

    Full Text Available Biochemistry underlays many subjects taught in high school but most teacherslack enough biochemical bases to explore them properly. To investigate their alternativeconceptions we have applied the distance course Biochemistry of Drugs to public schoolteachers, with class load of 30 hours and six modules: Statistics and basic concepts;Marijuana; Tobacco; Inhalants; Alcohol; Legalization vs Criminalization. The conceptionswere analyzed through the course records and the most important was the lack ofknowledge on the protein chemical structure, which impaired the comprehension ofproposed molecular mechanisms (involving receptors, neurotransmitters, enzymeinhibition, etc.. Several interventions promoted the overcoming of many misconceptionsas detected by written tests on chemical nature of involved compounds; neurotransmissionmechanism and the role of drugs in neurotransmission. Among 63 questions only 10 hadless than 50% correct answers. The teachers’ performances were impaired by readingdifficulties and poor scientific background that difficult their distinction of facts and scientificmodels from common sense or personal opinion. The teachers’ and the course staffevaluations were highly positive. Most of them declared that their knowledge was amplifiedand that they would recommend this course to colleagues. They also were favorablysurprised with the deep level of the topics, the demanded dedication and the fact that thecourse was addressed to themselves instead of to their students.

  20. RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing.

    Science.gov (United States)

    Maatz, Henrike; Jens, Marvin; Liss, Martin; Schafer, Sebastian; Heinig, Matthias; Kirchner, Marieluise; Adami, Eleonora; Rintisch, Carola; Dauksaite, Vita; Radke, Michael H; Selbach, Matthias; Barton, Paul J R; Cook, Stuart A; Rajewsky, Nikolaus; Gotthardt, Michael; Landthaler, Markus; Hubner, Norbert

    2014-08-01

    Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure. PMID:24960161

  1. A clinical approach to the nutritional care process in protein-energy wasting hemodialysis patients

    OpenAIRE

    Mar Ruperto; Sánchez-Muniz, Francisco J; Guillermina Barril

    2014-01-01

    Introduction: Malnutrition/wasting/cachexia are complex-disease conditions that frequently remain undiagnosed and/or untreated in up to 75% of prevalent hemodialysis (HD) patients. The nutrition care process (NCP) based on assessment, diagnosis, intervention and monitoring of nutritional status is a systematic method that nutrition professionals use to make decisions in clinical practice. Objective: This review examines from a clinical-nutritional practice point of view: a) nutritional status...

  2. Perilipin-mediated lipid droplet formation in adipocytes promotes sterol regulatory element-binding protein-1 processing and triacylglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

    Full Text Available Sterol regulatory element-binding protein-1 (SREBP-1 has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ enhances perilipin (plin gene expression, resulting in generating lipid droplets (LDs to store triacylglycerol (TAG in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER, alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

  3. A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

    Directory of Open Access Journals (Sweden)

    Maria Cesarina Abete

    2013-04-01

    Full Text Available Lifting of the ban on the use of processed animal proteins (PAPs from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR, which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork; one feed sample certified by the European reference laboratory on animal proteins (EURL AP in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

  4. The adaptive metabolic response involves specific protein glutathionylation during the filamentation process in the pathogen Candida albicans.

    Science.gov (United States)

    Gergondey, R; Garcia, C; Serre, V; Camadro, J M; Auchère, F

    2016-07-01

    Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition. Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Isocitrate lyase inactivation in the hyphal forms was reversed by glutaredoxin treatment, in agreement with a glutathionylation process, which was confirmed by proteomic data showing the binding of one glutathione molecule to the enzyme (data are available via ProteomeXchange with identifier PXD003685). We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and were related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen. PMID:27083931

  5. Functional and Physical Properties of Bovine Plasma Proteins as a Function of Processing and pH, Application in a Food Formulation

    Directory of Open Access Journals (Sweden)

    Mercedes Campderros

    2010-09-01

    Full Text Available The combined effects of ultrafiltration and freeze drying stages, and the incorporation of a stabilizing agent on selected functional and physical properties of bovine plasma proteins, were evaluated in the pH range of 3.0-9.0. The raw material was also characterized and was compared with the processed one. The results show that the process had a positive effect on solubility, emulsion capacity and emulsion stability in all the pH range, while the foaming capacity was similar to the raw material, having a better foam stability. The content of salts and denatured proteins was reduced in the processed plasma. This product was used in the formulation of a minced meat, which had a high stability and adequate moisture, improving the consistence according to the sensorial analysis. Therefore, the use of processed proteins in formulation of food products may be enhanced, and a higher added-value protein can be obtained.

  6. The Chemical Basis for the Origin of the Genetic Code and the Process of Protein Synthesis

    Science.gov (United States)

    Lacey, James C., Jr.

    1990-01-01

    A model for the origin of protein synthesis. The essential features of the model are that 5'-AMP and perhaps other monoribonucleotides can serve as catalysts for the selective synthesis of L-based peptides. A unique set of characteristics of 5'-AMP is responsible for the selective catalysts and these characteristics are described in detail. The model involves the formation of diesters as intermediates and selectivity for use of the L-isomer occurs principally at the step of forming the diester. However, in the formation of acetyl phenylalanine-AMP monoester there is a selectivity for esterification by the D-isomer. Data showing this selectivity is presented. This selectivity for D-isomer disappears after the first step. The identity was confirmed of all four of possible diesters of acetyl-D- and -L phenylaline with 5'-AMP by nuclear magnetic resonance (NMR). The data using flourescence and NMR show the Trp ring can associate with the adenine ring more strongly when the D-isomer is in the 2' position than it can when in the 3' position. These same data also suggest a molecular mechanisim for the faster esterificaton of 5'-AMP by acetyl-D-phenylaline. Some new data is also presented on the possible structure of the 2' isomer of acetyl-D-tryptophan-AMP monoester. The HPLC elution times of all four possible acetyl diphenylalanine esters of 5'-AMP were studied, these peptidyl esters will be products in the studies of peptide formation on the ribose of 5'-AMP. Other studies were on the rate of synthesis and the identity of the product when producing 3'Ac-Phe-2'tBOC-Phe-AMP diester. HPLC purification and identification of this product were accomplished.

  7. Effect of simulated processing on the antioxidant capacity and in vitro protein digestion of fruit juice-milk beverage model systems.

    Science.gov (United States)

    He, Zhiyong; Yuan, Bo; Zeng, Maomao; Tao, Guanjun; Chen, Jie

    2015-05-15

    The effects of simulated processing (pH adjustment and thermal treatment) on the antioxidant capacity and in vitro protein digestion of fruit juice-milk beverage (FJMB) models consisting of whey protein (WP), and chlorogenic acid (CHA) or catechin (CAT) were investigated. Results indicated that CAT was more susceptible to processing than CHA, and showed a significant (p 0.05) by pasteurization, whereas sterilization initially accelerated WP digestion but did not change its overall digestibility. PMID:25577106

  8. Biosynthesis of intestinal microvillar proteins. Further characterization of the intracellular processing and transport

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1984-01-01

    The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [35S]methionine. The expression of the microvilla...... presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N-linked carbohydrate....

  9. Protein kinase C-α mediates TNF release process in RBL-2H3 mast cells

    OpenAIRE

    Abdel-Raheem, Ihab T; Hide, Izumi; Yanase, Yuhki; Shigemoto-Mogami, Yukari; SAKAI, Norio; Shirai, Yasuhito; Saito, Naoaki; Hamada, Farid M; El-Mahdy, Nagh A; Elsisy, Alaa El-Din E; Sokar, Samya S; Nakata, Yoshihiro

    2005-01-01

    To clarify the mechanism of mast cell TNF secretion, especially its release process after being produced, we utilized an antiallergic drug, azelastine (4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4-yl)-1-(2H)- phthalazinone), which has been reported to inhibit TNF release without affecting its production in ionomycin-stimulated RBL-2H3 cells.Such inhibition was associated with the suppression of an ionomycin-induced increase in membrane-associated PKC activity rather than the suppressi...

  10. Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

    OpenAIRE

    Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

    2014-01-01

    The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein-protein and protein-lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane...

  11. Reduced amyloidogenic processing of the amyloid beta-protein precursor by the small-molecule Differentiation Inducing Factor-1.

    Science.gov (United States)

    Myre, Michael A; Washicosky, Kevin; Moir, Robert D; Tesco, Giuseppina; Tanzi, Rudolph E; Wasco, Wilma

    2009-04-01

    The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

  12. Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

    Science.gov (United States)

    Myre, Michael A.; Washicosky, Kevin; Moir, Robert D.; Tesco, Giuseppina; Tanzi, Rudolph E.; Wasco, Wilma

    2013-01-01

    The detection of cell cycle proteins in Alzheimer’s disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

  13. Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

    Energy Technology Data Exchange (ETDEWEB)

    Ugai, Hideyo; Dobbins, George C.; Wang, Minghui [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Le, Long P. [Massachusetts General Hospital, Pathology Service, 55 Fruit St.-GRJ 249, Boston, MA 02114 (United States); Matthews, David A. [School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, Bristol BS8 1TD (United Kingdom); Curiel, David T., E-mail: dcuriel@radonc.wustl.edu [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2012-10-25

    Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

  14. Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Teh, Thomas K H; Toh, Siew-Lok; Goh, James C H, E-mail: dosgohj@nus.edu.s, E-mail: dostkh@nus.edu.s, E-mail: bietohsl@nus.edu.s [Division of Bioengineering, National University of Singapore (Singapore)

    2010-06-01

    In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibroin structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 {sup 0}C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS-PAGE), respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS-PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 {sup 0}C.

  15. Circadian proteins CLOCK and BMAL1 in the chromatoid body, a RNA processing granule of male germ cells.

    Directory of Open Access Journals (Sweden)

    Rita L Peruquetti

    Full Text Available Spermatogenesis is a complex differentiation process that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells. RNA nuclear and cytoplasmic bodies appear to be critical for the progress of spermatogenesis. The chromatoid body (CB is a cytoplasmic organelle playing an important role in RNA post-transcriptional and translation regulation during the late steps of germ cell differentiation. The CB is also important for fertility determination since mutations of genes encoding its components cause infertility by spermatogenesis arrest. Targeted ablation of the Bmal1 and Clock genes, which encode central regulators of the circadian clock also result in fertility defects caused by problems other than spermatogenesis alterations. We show that the circadian proteins CLOCK and BMAL1 are localized in the CB in a stage-specific manner of germ cells. Both BMAL1 and CLOCK proteins physically interact with the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog, a hallmark component of the CB. BMAL1 is differentially expressed during the spermatogenic cycle of seminiferous tubules, and Bmal1 and Clock deficient mice display significant CB morphological alterations due to BMAL1 ablation or low expression. These findings suggest that both BMAL1 and CLOCK contribute to CB assembly and physiology, raising questions on the role of the circadian clock in reproduction and on the molecular function that CLOCK and BMAL1 could potentially have in the CB assembly and physiology.

  16. A clinical approach to the nutritional care process in protein-energy wasting hemodialysis patients

    Directory of Open Access Journals (Sweden)

    Mar Ruperto

    2014-04-01

    Full Text Available Introduction: Malnutrition/wasting/cachexia are complex-disease conditions that frequently remain undiagnosed and/or untreated in up to 75% of prevalent hemodialysis (HD patients. The nutrition care process (NCP based on assessment, diagnosis, intervention and monitoring of nutritional status is a systematic method that nutrition professionals use to make decisions in clinical practice. Objective: This review examines from a clinical-nutritional practice point of view: a nutritional status as a mortality causative factor; b phenotypic characteristics of malnutri-tion/wasting/cachexia, and c current trends of NCP with special emphasis on nutritional support and novel nutrient and pharmacologic adjunctive therapies in HD patients. Method: A literature review was conducted using the Pubmed, Science Direct, Scielo, Scopus, and Medline electronic scientific basis. Studies which assessing nutritional status and nutritional support published from 1990 to 2013 in HD patients were included and discussed. Results: From all the epidemiological data analyzed, NCP was the suggested method for identifying malnut rition/ wasting or cachexia in clinical practice. Nutrition support as an unimodal therapy was not completely able to reverse wasting in HD patients. Novel experimental therapeutic strategies including the use of appetite stimulants, ghrelin agonist, MC4-R antagonists, anabolic steroids, anti-inflammatory drugs, cholecalciferol, and other components are still under clinical evaluation. Conclusion: Nutritional status is a strong predictor of morbidity and mortality in HD patients. The terms called malnutrition, wasting and cachexia have different nutritional therapeutics implications. The NCP is a necessary tool for assessing and monitoring nutritional status in the current clinical practice. Novel pharmacological therapies or specific nutrient supplementation interventions studies are required.

  17. Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ngo, Hien-Ping; Hickson, Ian D

    2007-01-01

    CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination...... formation of MMS-induced HRR intermediates (X-molecules) arising during replication in sgs1 cells, mutation of SHU genes attenuated the level of these structures. Similar findings were also observed in shu1 cells in which Rmi1 or Top3 function was impaired. We propose a model in which the Shu proteins act...... in HRR to promote the formation of HRR intermediates that are processed by the Sgs1-Rmi1-Top3 complex....

  18. Changes in protein structures to improve the rheology and texture of reduced-fat sausages using high pressure processing.

    Science.gov (United States)

    Yang, Huijuan; Khan, Muhammad Ammar; Yu, Xiaobo; Zheng, Haibo; Han, Minyi; Xu, Xinglian; Zhou, Guanghong

    2016-11-01

    This study investigated the role of high-pressure processing (HPP) for improving the functional properties of meat batters and the textural properties of reduced-fat sausages. Application of 200MPa pressure at 10°C for 2min to pork batters containing various fat contents (0-30%) affected their rheological properties, cooking losses, color, textual properties and their protein imaging. The results revealed that both application of 200MPa and increasing fat content decreased cooking loss, as well as improved the textural and rheological properties. Cooking losses, texture and sensory evaluation of 200MPa treated sausages having 20% fat were similar to those of the 0.1MPa treated sausages having 30% fat. Principal component analysis revealed that certain quality attributes were affected differently by the levels of fat addition and by HPP. These findings indicated the potential of HPP for improving yield and texture of emulsion-type sausages having reduced fat contents. PMID:27288900

  19. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham.

    Science.gov (United States)

    Gallego, Marta; Mora, Leticia; Concepción Aristoy, M; Toldrá, Fidel

    2016-04-01

    The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham. PMID:26593512

  20. Simultaneous recovery and purification of rice protein and phosphorus compounds from full-fat and defatted rice bran with organic solvent-free process.

    Science.gov (United States)

    Watanabe, Masanori; Maeda, Isamu; Koyama, Masahiro; Nakamura, Kozo; Sasano, Kazuo

    2015-02-01

    We studied a process that enables simultaneous recovery of protein and phosphorus compounds from rice bran. Phosphorus substances in full-fat and defatted rice bran such as phytic acid and inorganic ions were solubilized under acidic conditions in the first step. After that, inorganic and/or organic phosphate salts were recovered in insoluble form under weak alkaline conditions. Furthermore, protein fractions obtained after phosphorus compounds had been removed were solubilized under alkaline conditions. After solubilization, protein fractions with high content were recovered by isoelectric precipitation (IP) followed by electrolyzed water treatment (EWT). The highest protein content (52.3 w/w%) was attained when machine defatted rice bran was treated through the process. Energy-dispersive X-ray spectroscopy (EDX) and inductively coupled plasma atomic emission spectrometry (ICP-AES) analyses demonstrated efficient desalting from the protein fractions by EWT and higher phosphorus contents (15.1-16.4 w/w% P) in the phosphorus fractions compared with commercial phosphate rock. In addition, no heavy metal ions in either protein or phosphorus fractions were detected. These results suggest that the newly developed process is suitable for practical recovery of highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants. PMID:25174654

  1. Effects of processing moisture on the physical properties and in vitro digestibility of starch and protein in extruded brown rice and pinto bean composite flours.

    Science.gov (United States)

    Sumargo, Franklin; Gulati, Paridhi; Weier, Steven A; Clarke, Jennifer; Rose, Devin J

    2016-11-15

    The influence of pinto bean flour and processing moisture on the physical properties and in vitro digestibility of rice-bean extrudates has been investigated. Brown rice: pinto bean flour (0%, 15%, 30%, and 45% bean flour) were extruded under 5 moisture conditions (17.2%, 18.1%, 18.3%, 19.5%, and 20.1%). Physical properties [bulk density, unit density, radial expansion, axial expansion, overall expansion, specific volume, hardness, color, water solubility index, and water absorption index] and in vitro starch and protein digestibilities were determined. Increasing bean flour and processing moisture increased density and hardness while decreasing expansion. Rapidly digestible starch decreased and resistant starch increased as bean substitution and processing moisture increased. In vitro protein digestibility increased with increasing bean flour or with decreasing processing moisture. Incorporating bean flour into extruded snacks can negatively affect physical attributes (hardness, density, and expansion) while positively affecting in vitro starch (decrease) and protein (increase) digestibilities. PMID:27283689

  2. Molecular spectroscopic investigation on fractionation-induced changes on biomacromolecule of co-products from bioethanol processing to explore protein metabolism in ruminants

    Science.gov (United States)

    Zhang, Xuewei; Yan, Xiaogang; Beltranena, Eduardo; Yu, Peiqiang

    2014-03-01

    Fractionation processing is an efficient technology which is capable to redesign/redevelop a new food or feed product with a specified chemical and nutrient profile. This processing technique was able to produce four different fractions (called "A", "B", "C", "D" fractions/treatments) with different nutrient profile form a co-product of bioethanol processing [wheat dried distillers grains with soluble (DDGS)]. To date, there is no study on the effect of fractionation processing on inherent molecular structure of different fractions and how the processing-induced structural change affect the metabolic characteristics of protein and nutrient availability. The objectives of this experiment were to: (1) investigate the effect of fractionation processing on changes of protein functional groups (amide I, amide II, and their ratio) and molecular structure (modeled α-helix, β-sheet, and their ratio), and (2) study the relationship between the fractionation processing-induced changes of protein molecular structure and nutrients availability as well as the metabolic characteristics of protein. The hypothesis of this study was that the fractionation processing changes the molecular structure and such changes affect the metabolic characteristics of protein. The protein molecular structure spectral profile of the fractions A, B, C and D were identified by Fourier-transform infrared attenuated total reflection spectroscopy (FT/IR-ATR). The results showed that the fractionation processing significantly affected the protein molecular spectral profiles. The differences in amide I to amide II peak area and height ratios were strongly significant (P < 0.01) among the treatment fractions, ranging from 4.98 to 6.33 and 3.28 to 4.00, respectively. The difference in the modeled protein α-helix to β-sheet ratio was also strongly significant (P < 0.01) among the treatment fractions. Multivariate molecular spectral analysis with cluster (CLA) and principal component analyses (PCA

  3. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding.

    Science.gov (United States)

    Boddey, Justin A; O'Neill, Matthew T; Lopaticki, Sash; Carvalho, Teresa G; Hodder, Anthony N; Nebl, Thomas; Wawra, Stephan; van West, Pieter; Ebrahimzadeh, Zeinab; Richard, Dave; Flemming, Sven; Spielmann, Tobias; Przyborski, Jude; Babon, Jeff J; Cowman, Alan F

    2016-01-01

    Plasmodium falciparum exports proteins into erythrocytes using the Plasmodium export element (PEXEL) motif, which is cleaved in the endoplasmic reticulum (ER) by plasmepsin V (PMV). A recent study reported that phosphatidylinositol-3-phosphate (PI(3)P) concentrated in the ER binds to PEXEL motifs and is required for export independent of PMV, and that PEXEL motifs are functionally interchangeable with RxLR motifs of oomycete effectors. Here we show that the PEXEL does not bind PI(3)P, and that this lipid is not concentrated in the ER. We find that RxLR motifs cannot mediate export in P. falciparum. Parasites expressing a mutated version of KAHRP, with the PEXEL motif repositioned near the signal sequence, prevented PMV cleavage. This mutant possessed the putative PI(3)P-binding residues but is not exported. Reinstatement of PEXEL to its original location restores processing by PMV and export. These results challenge the PI(3)P hypothesis and provide evidence that PEXEL position is conserved for co-translational processing and export. PMID:26832821

  4. Domestication process of the goat revealed by an analysis of the nearly complete mitochondrial protein-encoding genes.

    Directory of Open Access Journals (Sweden)

    Koh Nomura

    Full Text Available Goats (Capra hircus are one of the oldest domesticated species, and they are kept all over the world as an essential resource for meat, milk, and fiber. Although recent archeological and molecular biological studies suggested that they originated in West Asia, their domestication processes such as the timing of population expansion and the dynamics of their selection pressures are little known. With the aim of addressing these issues, the nearly complete mitochondrial protein-encoding genes were determined from East, Southeast, and South Asian populations. Our coalescent time estimations suggest that the timing of their major population expansions was in the Late Pleistocene and significantly predates the beginning of their domestication in the Neolithic era (≈10,000 years ago. The ω (ratio of non-synonymous rate/synonymous substitution rate for each lineage was also estimated. We found that the ω of the globally distributed haplogroup A which is inherited by more than 90% of goats examined, turned out to be extremely low, suggesting that they are under severe selection pressure probably due to their large population size. Conversely, the ω of the Asian-specific haplogroup B inherited by about 5% of goats was relatively high. Although recent molecular studies suggest that domestication of animals may tend to relax selective constraints, the opposite pattern observed in our goat mitochondrial genome data indicates the process of domestication is more complex than may be presently appreciated and cannot be explained only by a simple relaxation model.

  5. Multiple RNA processing defects and impaired chloroplast function in plants deficient in the organellar protein-only RNase P enzyme.

    Directory of Open Access Journals (Sweden)

    Wenbin Zhou

    Full Text Available Transfer RNA (tRNA precursors undergo endoribonucleolytic processing of their 5' and 3' ends. 5' cleavage of the precursor transcript is performed by ribonuclease P (RNase P. While in most organisms RNase P is a ribonucleoprotein that harbors a catalytically active RNA component, human mitochondria and the chloroplasts (plastids and mitochondria of seed plants possess protein-only RNase P enzymes (PRORPs. The plant organellar PRORP (PRORP1 has been characterized to some extent in vitro and by transient gene silencing, but the molecular, phenotypic and physiological consequences of its down-regulation in stable transgenic plants have not been assessed. Here we have addressed the function of the dually targeted organellar PRORP enzyme in vivo by generating stably transformed Arabidopsis plants in which expression of the PRORP1 gene was suppressed by RNA interference (RNAi. PRORP1 knock-down lines show defects in photosynthesis, while mitochondrial respiration is not appreciably affected. In both plastids and mitochondria, the effects of PRORP1 knock-down on the processing of individual tRNA species are highly variable. The drastic reduction in the levels of mature plastid tRNA-Phe(GAA and tRNA-Arg(ACG suggests that these two tRNA species limit plastid gene expression in the PRORP1 mutants and, hence, are causally responsible for the mutant phenotype.

  6. Phytic acid, in vitro protein digestibility, dietary fiber, and minerals of pulses as influenced by processing methods.

    Science.gov (United States)

    Chitra, U; Singh, U; Rao, P V

    1996-06-01

    The objective of this project was to determine the effect of various types of processing on selected nutrition related parameters of commonly consumed Indian pulses and soybean. Germination reduced the phytic acid content of chickpea and pigeonpea seeds by over 60%, and that of mung bean, urd bean, and soybean by about 40%. Fermentation reduced phytic acid contents by 26-39% in all these legumes with the exception of pigeonpea in which it was reduced by more than 50%. Autoclaving and roasting were more effective in reducing phytic acid in chickpea and pigeonpea than in urd bean, mung bean, and soybean. Germination and fermentation greatly increased the in vitro protein digestibility (IVPD). IVPD was only slightly increased by roasting and autoclaving of all legumes. Germination and fermentation also remarkably decreased the total dietary fiber (TDF) in all legumes. Autoclaving and roasting resulted in slight increases in TDF values. All the processing treatments had little effect on calcium, magnesium and iron contents. PMID:8983057

  7. Role of the NH2-terminal Membrane Spanning Domain of Multidrug Resistance Protein 1/ABCC1 in Protein Processing and TraffickingD⃞

    OpenAIRE

    Westlake, Christopher J.; Cole, Susan P.C.; Deeley, Roger G.

    2005-01-01

    Multidrug resistance protein (MRP)1/ABCC1 transports organic anionic conjugates and confers resistance to cytotoxic xenobiotics. In addition to two membrane spanning domains (MSDs) typical of most ATP-binding cassette (ABC) transporters, MRP1 has a third MSD (MSD0) of unknown function. Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effect on function, nor does it prevent MRP1 from trafficking to basolateral membranes in polarized cells. However, we find that inde...

  8. Effects of buffer additives and thermal processing methods on the solubility of shrimp (Penaeus monodon) proteins and the immunoreactivity of its major allergen.

    Science.gov (United States)

    Lasekan, Adeseye O; Nayak, Balunkeswar

    2016-06-01

    This study examines the potential of two buffer additives (Tween 20 and DTT) to improve the solubility of proteins from shrimp subjected to different heat treatments and the allergenicity of tropomyosin in the extracts. The concentration of soluble proteins extracted by all the buffers from processed shrimp was significantly reduced compared with untreated samples. The concentration of total soluble proteins from heat treated shrimp increased significantly when phosphate buffer containing both surfactant and reducing agent was used as the extraction buffer. However, the concentrations of heat-stable proteins in the buffers were mostly similar. The electrophoretic profile of extracted proteins showed that tropomyosin is very stable under the different heat treatment methods used in this study except for high pressure steaming where the intensity of tropomyosin band was reduced. Competitive inhibition ELISA showed that high pressure steaming reduced the allergenicity of tropomyosin compared with other heat treatments methods. PMID:26830572

  9. Role of the NH2-terminal membrane spanning domain of multidrug resistance protein 1/ABCC1 in protein processing and trafficking.

    Science.gov (United States)

    Westlake, Christopher J; Cole, Susan P C; Deeley, Roger G

    2005-05-01

    Multidrug resistance protein (MRP)1/ABCC1 transports organic anionic conjugates and confers resistance to cytotoxic xenobiotics. In addition to two membrane spanning domains (MSDs) typical of most ATP-binding cassette (ABC) transporters, MRP1 has a third MSD (MSD0) of unknown function. Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effect on function, nor does it prevent MRP1 from trafficking to basolateral membranes in polarized cells. However, we find that independent of cell type, the truncated protein accumulates in early/recycling endosomes. Using a real-time internalization assay, we demonstrate that MSD0 is important for MRP1 retention in, or recycling to, the plasma membrane. We also show that MSD0 traffics independently to the cell surface and promotes membrane localization of the core-region of MRP1 when the two protein fragments are coexpressed. Finally, we demonstrate that MSD0 becomes essential for trafficking of MRP1 when the COOH-terminal region of the protein is mutated. These studies demonstrate that MSD0 and the COOH-terminal region contain redundant trafficking signals, which only become essential when one or the other region is missing or is mutated. These data explain apparent differences in the trafficking requirement for MSD0 and the COOH-terminal region of MRP1 compared with other ABCC proteins. PMID:15772158

  10. THE CHANGE OF TOTAL PROTEIN FRACTION OF MUSCLE TISSUE OF PORK WITH BIO- AND PHYSICO-CHEMICAL SPECIFIC IN THE PROCESS OF COOKING AT DIFFERENT TEMPERATURES

    Directory of Open Access Journals (Sweden)

    O. Shalimova

    2012-03-01

    Full Text Available The character of changes in total protein fraction of muscle tissue of pork with PSE defects in the process of cooking at temperatures ranging from 40 to 72 g.C in steps of 2 g.C is investigated. Our studies have revealed differences in the change of state the total fraction of muscle proteins with defects PSE pork during cooking.

  11. CRISPR-Cas Systems in the Cyanobacterium Synechocystis sp. PCC6803 Exhibit Distinct Processing Pathways Involving at Least Two Cas6 and a Cmr2 Protein

    OpenAIRE

    Scholz, Ingeborg; Lange, Sita J.; Hein, Stephanie; Wolfgang R Hess; Backofen, Rolf

    2013-01-01

    The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats – CRISPR associated proteins) system provides adaptive immunity in archaea and bacteria. A hallmark of CRISPR-Cas is the involvement of short crRNAs that guide associated proteins in the destruction of invading DNA or RNA. We present three fundamentally distinct processing pathways in the cyanobacterium Synechocystis sp. PCC6803 for a subtype I-D (CRISPR1), and two type III systems (CRISPR2 and CRISPR3), which are locate...

  12. A novel one-pot process for near-net-shape fabrication of open-porous resorbable hydroxyapatite/protein composites and in vivo assessment

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Berit, E-mail: beritm@uni-bremen.de [University of Bremen, Advanced Ceramics, Am Biologischen Garten 2, 28359 Bremen (Germany); Koch, Dietmar, E-mail: dietmar.koch@dlr.de [German Aerospace Center, Ceramic Composite Structures, Pfaffenwaldring 38-40, 70569 Stuttgart (Germany); Lutz, Rainer, E-mail: rainer.lutz@uk-erlangen.de [University of Erlangen-Nuremberg, Department of Oral and Maxillofacial Surgery, Glueckstrasse 11, 91054 Erlangen (Germany); Schlegel, Karl A., E-mail: andreas.schlegel@uk-erlangen.de [University of Erlangen-Nuremberg, Department of Oral and Maxillofacial Surgery, Glueckstrasse 11, 91054 Erlangen (Germany); Treccani, Laura, E-mail: treccani@uni-bremen.de [University of Bremen, Advanced Ceramics, Am Biologischen Garten 2, 28359 Bremen (Germany); Rezwan, Kurosch, E-mail: krezwan@uni-bremen.de [University of Bremen, Advanced Ceramics, Am Biologischen Garten 2, 28359 Bremen (Germany)

    2014-09-01

    We present a mild one-pot freeze gelation process for fabricating near-net, complex-shaped hydroxyapatite scaffolds and to directly incorporate active proteins during scaffold processing. In particular, the direct protein incorporation enables a simultaneous adjustment and control of scaffold microstructure, porosity, resorbability and enhancement of initial mechanical and handling stability. Two proteins, serum albumin and lysozyme, are selected and their effect on scaffold stability and microstructure investigated by biaxial strength tests, electron microscopy, and mercury intrusion porosimetry. The resulting hydroxyapatite/protein composites feature adjustable porosities from 50% to 70% and a mechanical strength ranging from 2 to 6 MPa comparable to that of human spongiosa without any sintering step. Scaffold degradation behaviour and protein release are assessed by in vitro studies. A preliminary in vivo assessment of scaffold biocompatibility and resorption behaviour in adult domestic pigs is discussed. After implantation, composites were resorbed up to 50% after only 4 weeks and up to 65% after 8 weeks. In addition, 14% new bone formation after 4 weeks and 37% after 8 weeks were detected. All these investigations demonstrate the outstanding suitability of the one-pot-process to create, in a customisable and reliable way, biocompatible scaffolds with sufficient mechanical strength for handling and surgical insertion, and for potential use as biodegradable bone substitutes and versatile platform for local drug delivery. - Highlights: • We present a one-pot process for directly incorporating protein into HAp scaffolds. • The effect of two model proteins, BSA and LSZ, on scaffold properties is analysed. • HAp/protein scaffolds feature a mechanical strength comparable to human spongiosa. • BSA incorporation in scaffolds leads to strength increase despite porosity increment. • New bone formation in-vivo exceeds established xenograft bone substitutes.

  13. A novel one-pot process for near-net-shape fabrication of open-porous resorbable hydroxyapatite/protein composites and in vivo assessment

    International Nuclear Information System (INIS)

    We present a mild one-pot freeze gelation process for fabricating near-net, complex-shaped hydroxyapatite scaffolds and to directly incorporate active proteins during scaffold processing. In particular, the direct protein incorporation enables a simultaneous adjustment and control of scaffold microstructure, porosity, resorbability and enhancement of initial mechanical and handling stability. Two proteins, serum albumin and lysozyme, are selected and their effect on scaffold stability and microstructure investigated by biaxial strength tests, electron microscopy, and mercury intrusion porosimetry. The resulting hydroxyapatite/protein composites feature adjustable porosities from 50% to 70% and a mechanical strength ranging from 2 to 6 MPa comparable to that of human spongiosa without any sintering step. Scaffold degradation behaviour and protein release are assessed by in vitro studies. A preliminary in vivo assessment of scaffold biocompatibility and resorption behaviour in adult domestic pigs is discussed. After implantation, composites were resorbed up to 50% after only 4 weeks and up to 65% after 8 weeks. In addition, 14% new bone formation after 4 weeks and 37% after 8 weeks were detected. All these investigations demonstrate the outstanding suitability of the one-pot-process to create, in a customisable and reliable way, biocompatible scaffolds with sufficient mechanical strength for handling and surgical insertion, and for potential use as biodegradable bone substitutes and versatile platform for local drug delivery. - Highlights: • We present a one-pot process for directly incorporating protein into HAp scaffolds. • The effect of two model proteins, BSA and LSZ, on scaffold properties is analysed. • HAp/protein scaffolds feature a mechanical strength comparable to human spongiosa. • BSA incorporation in scaffolds leads to strength increase despite porosity increment. • New bone formation in-vivo exceeds established xenograft bone substitutes

  14. The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

    Energy Technology Data Exchange (ETDEWEB)

    Gemelli, Claudia, E-mail: claudia.gemelli@unimore.it [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy); Center for Regenerative Medicine, University of Modena and Reggio Emilia, Via Gottardi 100, 41125 Modena (Italy); Martello, Andrea; Montanari, Monica; Zanocco Marani, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis [Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy)

    2013-12-10

    Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1 kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 – VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling. - Highlights: • ORM1 is a Vitamin D primary response gene. • VD and its receptor VDR are involved in the de-activation process mediated by human resident macrophages. • The signaling pathway VD-VDR-ORM1 plays an important role in the control of macrophage de-activation process. • ORM1 may be defined as a signaling molecule implicated in the maintenance of tissue homeostasis and remodeling.

  15. The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

    International Nuclear Information System (INIS)

    Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1 kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 – VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling. - Highlights: • ORM1 is a Vitamin D primary response gene. • VD and its receptor VDR are involved in the de-activation process mediated by human resident macrophages. • The signaling pathway VD-VDR-ORM1 plays an important role in the control of macrophage de-activation process. • ORM1 may be defined as a signaling molecule implicated in the maintenance of tissue homeostasis and remodeling

  16. Transamidation of gluten proteins during the bread-making process of wheat flour to produce breads with less immunoreactive gluten.

    Science.gov (United States)

    Heredia-Sandoval, Nina Gisella; Islas-Rubio, Alma Rosa; Cabrera-Chávez, Francisco; Calderón de la Barca, Ana María

    2014-08-01

    Due to an increasing incidence of celiac disease (CD) and other gluten-related disorders, different gluten-free breads have been developed using starches and additives as a substitute for gluten. Thus, patients miss not only the taste and aroma of wheat bread but also risk their sensitive intestines. Therefore, modifying gluten to avoid an immune response in CD and its application to baking is in progress. The aim of the study was to enzymatically modify gluten on wheat flour, during bread-making avoiding the use of additives, to reduce immunoreactivity, preserving its properties. Microbial transglutaminase (mTG) or chymotrypsin (ChT) was used to bind lysine or valine to gluten proteins in a model system. The best conditions were directly applied to wheat flour for bread-making with and without punching at 45 min. Subsequently, the rheological properties of the doughs, specific volume of the loaves, immunoreactive gluten content and modification of the extracted proteins were evaluated. ChT-treated breads presented a better appearance with a more homogeneous crumb, higher specific volume values (3.34-4.25 cm(3) g(-1)) and higher reactive gluten reduction (up to 71%) than the mTG-treated ones (1.23-2.66 cm(3) g(-1)) with only a 42% reactive gluten reduction. Thus, transpeptidation during bread-making is a promising technology, although it is necessary to improve the modification process to obtain the reactive gluten reduction required in breads for the treatment of CD patients and other gluten-related disorders. PMID:24917417

  17. FLASH, a pro-apoptotic protein involved in activation of caspase-8 is essential for 3′ end processing of histone pre-mRNAs

    OpenAIRE

    Yang, Xiao-cui; Burch, Brandon D.; Yan, Yan; Marzluff, William F.; Dominski, Zbigniew

    2009-01-01

    3′ end processing of histone pre-mRNA requires U7 snRNP, which binds downstream of the cleavage site and recruits the endonuclease CPSF-73. U7 snRNP contains a unique Sm ring in which the canonical SmD2 protein is replaced by Lsm11. We used the yeast two-hybrid system to identify binding partners of Lsm11 and selected the pro-apoptotic protein FLASH. Human FLASH interacts with Lsm11 in vitro and stimulates 3′ end processing of histone pre-mRNA in mammalian nuclear extracts. We also identified...

  18. Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Gasser Brigitte

    2006-12-01

    Full Text Available Abstract Background Secretion of heterologous proteins depends both on biomass concentration and on the specific product secretion rate, which in turn is not constant at varying specific growth rates. As fed batch processes usually do not maintain a steady state throughout the feed phase, it is not trivial to model and optimize such a process by mathematical means. Results We have developed a model for product accumulation in fed batch based on iterative calculation in Microsoft Excel spreadsheets, and used the Solver software to optimize the time course of the media feed in order to maximize the volumetric productivity. The optimum feed phase consisted of an exponential feed at maximum specific growth rate, followed by a phase with linearly increasing feed rate and consequently steadily decreasing specific growth rate. The latter phase could be modeled also by exact mathematical treatment by the calculus of variations, yielding the explicit shape of the growth function, however, with certain indeterminate parameters. To evaluate the latter, one needs a numerical optimum search algorithm. The explicit shape of the growth function provides additional evidence that the Excel model results in correct data. Experimental evaluation in two independent fed batch cultures resulted in a good correlation to the optimized model data, and a 2.2 fold improvement of the volumetric productivity. Conclusion The advantages of the procedure we describe here are the ease of use and the flexibility, applying software familiar to every scientist and engineer, and rapid calculation which makes predictions extremely easy, so that many options can be tested in silico quickly. Additional options like further biological and technological constraints or different functions for specific productivity and biomass yield can easily be integrated.

  19. [Effect of the extrusion process on the functional characteristics and protein quality of quinua (Chenopodium quinoa, Willd)].

    Science.gov (United States)

    Romero, A; Bacigalupo, A; Bressani, R

    1985-03-01

    In order to have available a human food of high nutritive value, and conscious of the protein quality of the quinua, as well as its carbohydrate, vitamin and mineral content, its behavior during the extrusion process was tested in the present study. To eliminate saponins, a simple method was developed which consisted of washing the seeds through an aluminum container, using a wooden stirrer. Seven treatments were studied: washed quinua, washed and cooked quinua, washed and expanded quinua No. 1 and No. 2, and washed and texturized quinua No. 1 and No. 2; casein was used as control. Biological evaluation trials were carried out in Holtzman rats, following the PER method. To detect the possible effects of the processed quinua on the experimental animals, hematological as well as histopathological studies of the vital organs were performed. A maximum PER of 2.43 was obtained for the texturized quinua, 2.16 for the expanded quinua, 2.6 for the cooked quinua, while the casein control yielded a PER of 3.00. The physico-chemical characteristics of the quinua flour were determined, as well as those of the expanded and texturized products. The product obtained was subjected to an organoleptic trial and it can be stated that the results obtained were satisfactory. The product can be consumed directly without major modifications and has an acceptable flavor. The nutritive value of quinua was not impaired; it compared favorably with the best diets recommended for the population, especially of those with a lower income. The results obtained in the present study suggest the possibility of increasing the nutritional value of the product, as well as its acceptability. PMID:3834873

  20. [Mutation process in the protein-coding genes of human mitochondrial genome in context of evolution of the genus].

    Science.gov (United States)

    Maliarchuk, B A

    2013-01-01

    The human mitochondrial genome, although it has a small size, is characterized by high level of variation, non-uniformly distributed in groups of nucleotide positions that differ in the degree of variability. Considering the mutation process in human mtDNA relative to the mitochondrial genomes of the genus Homo-neandertals, denisova hominin and other primate species, it appears that more than half (56.5%) variable positions in the human mtDNA protein-coding genes are characterized by back (reverse) mutations to the pre-H. sapiens state of mitochondrial genome. It has been found that hypervariable nucleotide positions show a minimal proportion of specific to H. sapiens mutations, and, conversely, a high proportion of mutations (both nucleotide and amino acid substitutions), leading to the loss of Homo-specific variants of polymorphisms. Most often, polymorphisms specific to H. sapiens arise in result of single forward mutations and disappear mainly due to multiple back mutations, including those in the mutational "hotspots". PMID:25509854

  1. Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy

    Science.gov (United States)

    Muñoz, Vanessa C.; Yefi, Claudia P.; Bustamante, Hianara A.; Barraza, Rafael R.; Tapia-Rojas, Cheril; Otth, Carola; Barrera, María José; González, Carlos; Mardones, Gonzalo A.; Inestrosa, Nibaldo C.; Burgos, Patricia V.

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo. PMID:26308941

  2. Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

    Science.gov (United States)

    Cavieres, Viviana A; González, Alexis; Muñoz, Vanessa C; Yefi, Claudia P; Bustamante, Hianara A; Barraza, Rafael R; Tapia-Rojas, Cheril; Otth, Carola; Barrera, María José; González, Carlos; Mardones, Gonzalo A; Inestrosa, Nibaldo C; Burgos, Patricia V

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo. PMID:26308941

  3. U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3′ end processing

    OpenAIRE

    Azzouz, Teldja N.; Gruber, Andreas; Schümperli, Daniel

    2005-01-01

    The 3' cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100....

  4. The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

    Science.gov (United States)

    de Lira, Nayara Patricia Vieira; Quaresma, Alexandre José Christino; Pauletti, Bianca Alves; Leme, Adriana Franco Paes; Benedetti, Celso Eduardo

    2012-01-01

    Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control. PMID:22384209

  5. The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U) RNA.

    Science.gov (United States)

    de Souza, Tiago Antonio; Soprano, Adriana Santos; de Lira, Nayara Patricia Vieira; Quaresma, Alexandre José Christino; Pauletti, Bianca Alves; Paes Leme, Adriana Franco; Benedetti, Celso Eduardo

    2012-01-01

    Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control. PMID:22384209

  6. A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.

    Science.gov (United States)

    Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen

    2013-11-29

    The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC. PMID:24139506

  7. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

    OpenAIRE

    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin

    2013-01-01

    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disord...

  8. Human Immunodeficiency Virus Protein Tat Induces Synapse Loss via a Reversible Process that is Distinct from Cell Death

    OpenAIRE

    Kim, Hee Jung; Martemyanov, Kirill A.; Thayer, Stanley A.

    2008-01-01

    Human immunodeficiency virus (HIV)-1 infection of the CNS produces changes in dendritic morphology that correlate with cognitive decline in patients with HIV-1 associated dementia (HAD). Here we investigated the effects of HIV-1 transactivator of transcription (Tat), a protein released by virus-infected cells, on synapses between hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein (PSD9...

  9. Effects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.

    OpenAIRE

    Vandevenne, Marylène; GASPARD, Genevieve; Belgsir, E. M.; Ramnath, M.; Cenatiempo, Y; Delneuville, Delphine; Dumoulin, Mireille; Frère, Jean-Marie; Matagne, André; Galleni, Moreno; Filee, P.

    2011-01-01

    Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. beta-Cyclodextrin (beta-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring ...

  10. Plasma membrane lipid-protein interactions affect signaling processes in sterol-biosynthesis mutants of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Henrik eZauber

    2014-03-01

    Full Text Available The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein-protein and protein-lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid-protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status.

  11. Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth12

    Science.gov (United States)

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-01-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein. PMID:23479509

  12. Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth

    Directory of Open Access Journals (Sweden)

    Daniela Trisciuoglio

    2013-03-01

    Full Text Available Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2 protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4 domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1 the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2 Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3 BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein.

  13. Removal of the BH4 domain from Bcl-2 protein triggers an autophagic process that impairs tumor growth.

    Science.gov (United States)

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-03-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein. PMID:23479509

  14. Influence of Processing on Dietary Fiber, Tannin and <i>in Vitro</i> Protein Digestibility of Pearl Millet

    OpenAIRE

    Florence Suma Pushparaj; Asna Urooj

    2011-01-01

    From the nutritional point of view, data on dietary fiber content, tannin and in vitro protein digestibility of processed millet is of importance, because millets are never eaten raw. Effects of commonly used traditional methods on dietary fiber, tannin content and %IVPD of two locally available pearl millet varieties (Kalukombu and Maharashtra Rabi Bajra) were investigated. The millet was subjected to various processing methods like milling (whole flour, semi refined flour and bran rich frac...

  15. Effects of Dietary Protein Concentration and L-carnitine on Growth, Processing Yield, and Body Composition of Channel X Blue Catfish Hybrids

    Science.gov (United States)

    A study was conducted in earthen ponds to evaluate effects of dietary protein concentration and L-carnitine supplementation on production and processing traits of channel catfish × blue catfish hybrids. Hybrid fingerlings, mean initial weight = 66 g, were stocked into 20, 0.04-ha earthen ponds at a...

  16. Effect of Different Processing Methods on Anti-Nutrients Content and Protein Quality of Improved Lupin (Lupinus Albus L. Cultivar Seeds

    Directory of Open Access Journals (Sweden)

    Mohamed Ahmed M. Omer

    2016-01-01

    Full Text Available Lupin seeds of genetically improved cultivar (Golo were subjected to different processing methods and investigated according to anti-nutritional factors content and protein quality. Results showed that tannin content of raw seeds was significantly increased in sprouted and debittered seeds before and after boiling but in fermented seeds it declined significantly. Phytate content was significantly decreased in all processed seeds with a significant reduction observed in germinated seeds. The reduction in Phytate as a result of processing was accompanied by a significant improvement in protein digestibility. The protein content of lupin seeds decreased in sprouted seeds and increased in fermented and debittered ones. Boiling of the seeds even the sprouted ones significantly increased the protein content compared to raw lupin seeds. In raw lupin seeds, globulins comprised the major fraction followed by glutelin. Debittered seeds characterized by high glutelin, fermented are characterized by high globulin while germinated characterized by both fractions. Most of the amino acids level was increased after processing of the seeds.

  17. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller;

    2008-01-01

    Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for c...

  18. Formation of long-lived radicals on proteins by radical transfer from heme enzymes--a common process?

    DEFF Research Database (Denmark)

    Ostdal, H; Andersen, H J; Davies, Michael Jonathan

    1999-01-01

    albumin via the heme edge of the peroxidase. In contrast, albumin radical formation by the HRP/H2O2/free tyrosine system was only marginally affected by proteolysis, consistent with free tyrosine phenoxyl radicals being the mediators of radical transfer, without significant protein-protein interaction...

  19. Comparative study of denaturation of whey protein isolate (WPI) in convective air drying and isothermal heat treatment processes.

    Science.gov (United States)

    Haque, M Amdadul; Aldred, Peter; Chen, Jie; Barrow, Colin J; Adhikari, Benu

    2013-11-15

    The extent and nature of denaturation of whey protein isolate (WPI) in convective air drying environments was measured and analysed using single droplet drying. A custom-built, single droplet drying instrument was used for this purpose. Single droplets having 5±0.1μl volume (initial droplet diameter 1.5±0.1mm) containing 10% (w/v) WPI were dried at air temperatures of 45, 65 and 80°C for 600s at constant air velocity of 0.5m/s. The extent and nature of denaturation of WPI in isothermal heat treatment processes was measured at 65 and 80°C for 600s and compared with those obtained from convective air drying. The extent of denaturation of WPI in a high hydrostatic pressure environment (600MPa for 600s) was also determined. The results showed that at the end of 600s of convective drying at 65°C the denaturation of WPI was 68.3%, while it was only 10.8% during isothermal heat treatment at the same medium temperature. When the medium temperature was maintained at 80°C, the denaturation loss of WPI was 90.0% and 68.7% during isothermal heat treatment and convective drying, respectively. The bovine serum albumin (BSA) fraction of WPI was found to be more stable in the convective drying conditions than β-lactoglobulin and α-lactalbumin, especially at longer drying times. The extent of denaturation of WPI in convective air drying (65 and 80°C) and isotheral heat treatment (80°C) for 600s was found to be higher than its denaturation in a high hydrostatic pressure environment at ambient temperature (600MPa for 600s). PMID:23790837

  20. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    Directory of Open Access Journals (Sweden)

    Zarbock Ralf

    2012-03-01

    Full Text Available Abstract Background Surfactant protein C (SP-C is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects. Methods SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide. Results Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. Conclusions We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy

  1. Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth12

    OpenAIRE

    Trisciuoglio, Daniela; De Luca, Teresa; Desideri, Marianna; Passeri, Daniela; Gabellini, Chiara; Scarpino, Stefania; Liang, Chengyu; Orlandi, Augusto; Del Bufalo, Donatella

    2013-01-01

    Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. I...

  2. Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

    Science.gov (United States)

    Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao

    2015-10-01

    This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed. PMID:26277627

  3. Effect of domestic processing on the cooking time, nutrients, antinutrients and in vitro protein digestibility of the African yambean (Sphenostylis stenocarpa).

    Science.gov (United States)

    Ene-obong, H N; Obizoba, I C

    1996-01-01

    The effects of processing (soaking, dehulling, fermentation and heat treatment) on the cooking time, protein, mineral, tannin, phytate and in vitro protein digestibility (IVPD) of the African yambean (AYB) were examined. The cooking time ranged from 90-155 minutes. Soaking reduced cooking time by about 50 percent. Soaking for 12 hours was the most appropriate to reduce cooking time, tannin and phytate levels. It improved in vitro protein digestibility (IVPD). Prolonged soaking (24 hours) decreased calcium (Ca) and iron (Fe) values by 19 percent and 35 percent, respectively. Dehulling showed that Ca, Fe, magnesium (Mg) and zinc (Zn) were concentrated in the seed coat of the AYB. The seeds soaked and dehulled retained Mg and Zn. Dehulling reduced tannin but had no significant effect on phytate and the IVPD of the AYB except for seeds soaked for 12 hours before dehulling. Soaking for 24 hours before dehulling significantly increased crude protein content by 16 percent (p roasting increased the IVPD by 8-11 percent. Fermentation had no effect on the crude protein, Ca, Fe, Mg and Zn but significantly reduced phytate content of the AYB. Fermentation had no advantage over heat treatment with respect to improving the in vitro protein digestibility of the AYB. PMID:9139303

  4. Bile acids modulate the Golgi membrane fission process via a protein kinase Ceta and protein kinase D-dependent pathway in colonic epithelial cells.

    Science.gov (United States)

    Byrne, Anne-Marie; Foran, Eilis; Sharma, Ruchika; Davies, Anthony; Mahon, Ciara; O'Sullivan, Jacintha; O'Donoghue, Diarmuid; Kelleher, Dermot; Long, Aideen

    2010-04-01

    Deoxycholic acid (DCA) is a secondary bile acid that modulates signalling pathways in epithelial cells. DCA has been implicated in pathogenesis of colon carcinoma, particularly by activation of the protein kinase C (PKC) pathway. Ursodeoxycholic acid (UDCA), a tertiary bile acid, has been observed to have chemopreventive effects. The aim of this study was to investigate the effect of DCA and UDCA on the subcellular localization and activity of PKCeta and its downstream effects on Golgi structure in a colon cancer cell model. PKCeta expression was localized to the Golgi in HCT116 colon cancer cells. DCA induced fragmentation of the Golgi in these cells following activation of PKCeta and its downstream effector protein kinase D (PKD). Pretreatment of cells with UDCA or a glucocorticoid, dexamethasone, inhibited DCA-induced PKCeta/PKD activation and Golgi fragmentation. Knockdown of glucocorticoid receptor (GR) expression using small interfering RNA or inhibition using the GR antagonist mifepristone attenuated the inhibitory effect of UDCA on Golgi fragmentation. Elevated serum and faecal levels of DCA have been previously reported in patients with ulcerative colitis (UC) and colon cancer. Analysis of Golgi architecture in vivo using tissue microarrays revealed Golgi fragmentation in UC and colorectal cancer tissue. We have demonstrated that DCA can disrupt the structure of the Golgi, an organelle critical for normal cell function. Inhibition of this DCA-induced Golgi fragmentation by UDCA was mediated via the GR. This represents a potential mechanism of observed chemopreventive effects of UDCA in benign and malignant disease of the colon. PMID:20093383

  5. Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-α protein (PMPCA) cause a severe mitochondrial disease.

    Science.gov (United States)

    Joshi, Mugdha; Anselm, Irina; Shi, Jiahai; Bale, Tejus A; Towne, Meghan; Schmitz-Abe, Klaus; Crowley, Laura; Giani, Felix C; Kazerounian, Shideh; Markianos, Kyriacos; Lidov, Hart G; Folkerth, Rebecca; Sankaran, Vijay G; Agrawal, Pankaj B

    2016-05-01

    We describe a large Lebanese family with two affected members, a young female proband and her male cousin, who had multisystem involvement including profound global developmental delay, severe hypotonia and weakness, respiratory insufficiency, blindness, and lactic acidemia-findings consistent with an underlying mitochondrial disorder. Whole-exome sequencing was performed on DNA from the proband and both parents. The proband and her cousin carried compound heterozygous mutations in the PMPCA gene that encodes for α-mitochondrial processing peptidase (α-MPP), a protein likely involved in the processing of mitochondrial proteins. The variants were located close to and postulated to affect the substrate binding glycine-rich loop of the α-MPP protein. Functional assays including immunofluorescence and western blot analysis on patient's fibroblasts revealed that these variants reduced α-MPP levels and impaired frataxin production and processing. We further determined that those defects could be rescued through the expression of exogenous wild-type PMPCA cDNA. Our findings link defective α-MPP protein to a severe mitochondrial disease. PMID:27148589

  6. Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor.

    OpenAIRE

    Peng, X; Mount, S M

    1995-01-01

    SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggera...

  7. Direct observation of the binding process between protein and quantum dots by in situ surface plasmon resonance measurements

    International Nuclear Information System (INIS)

    A layer-by-layer surface decoration technique has been developed to anchor quantum dots (QDs) onto a gold substrate and an in situ surface plasmon resonance technique has been used to study interactions between the QDs and different proteins. Direct observation of the binding of the protein onto the QDs and the kinetics of the adsorption and dissociation of different proteins on the QDs has been achieved. This would be helpful for the identification of particle-associated proteins and may offer a fundamental prerequisite for nanobiology, nanomedicine and nanotoxicology. The combination of the novel layer-by-layer surface modification method and in situ surface plasmon resonance would be powerful in studying biological systems such as DNA and cells.

  8. Direct observation of the binding process between protein and quantum dots by in situ surface plasmon resonance measurements

    Energy Technology Data Exchange (ETDEWEB)

    Xiao Qi; Zhou Bo; Tian Fangfang; Ge Yushu; Liu Xiaorong; Liu Yi [State Key Laboratory of Virology, College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Huang Shan; Guan Hongliang; He Zhike, E-mail: prof.liuyi@263.ne [Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China)

    2009-08-12

    A layer-by-layer surface decoration technique has been developed to anchor quantum dots (QDs) onto a gold substrate and an in situ surface plasmon resonance technique has been used to study interactions between the QDs and different proteins. Direct observation of the binding of the protein onto the QDs and the kinetics of the adsorption and dissociation of different proteins on the QDs has been achieved. This would be helpful for the identification of particle-associated proteins and may offer a fundamental prerequisite for nanobiology, nanomedicine and nanotoxicology. The combination of the novel layer-by-layer surface modification method and in situ surface plasmon resonance would be powerful in studying biological systems such as DNA and cells.

  9. Direct observation of the binding process between protein and quantum dots by in situ surface plasmon resonance measurements

    Science.gov (United States)

    Xiao, Qi; Zhou, Bo; Huang, Shan; Tian, Fangfang; Guan, Hongliang; Ge, Yushu; Liu, Xiaorong; He, Zhike; Liu, Yi

    2009-08-01

    A layer-by-layer surface decoration technique has been developed to anchor quantum dots (QDs) onto a gold substrate and an in situ surface plasmon resonance technique has been used to study interactions between the QDs and different proteins. Direct observation of the binding of the protein onto the QDs and the kinetics of the adsorption and dissociation of different proteins on the QDs has been achieved. This would be helpful for the identification of particle-associated proteins and may offer a fundamental prerequisite for nanobiology, nanomedicine and nanotoxicology. The combination of the novel layer-by-layer surface modification method and in situ surface plasmon resonance would be powerful in studying biological systems such as DNA and cells.

  10. Modeling of kinetics of the inducible protein complexes of the SOS system in bacteria E. coli which realize TLS process

    International Nuclear Information System (INIS)

    The mathematical model describing kinetics of the inducible genes of the protein complexes, formed during SOS response in bacteria Escherichia coli is developed. Within the bounds of developed approaches the auxiliary mathematical model describing changes in concentrations of the dimers, which are the components of final protein complexes, is developed. The solutions of both models are based on the experimental data concerning expression of the basic genes of the SOS system in bacteria Escherichia coli

  11. Research Advances on the Process and Application of Whey Protein%乳清蛋白及其加工利用的研究进展

    Institute of Scientific and Technical Information of China (English)

    陈静廷

    2013-01-01

    乳清蛋白作为优质的蛋白质来源,以其良好的功能特性受到了人们的广泛关注,其生产形式多样、应用范围广泛.本文主要综述了乳清蛋白的几种加工方法及其应用现状.%More attention was paid for the major properties of whey protein as a source of protein which was high quality and vaired forms of exploitation with a wide rang of application.This article reviewed several processing methods and the application of whey protein.

  12. Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor-β1 into a biologically active protein.

    Science.gov (United States)

    Wilbers, Ruud H P; Westerhof, Lotte B; van Raaij, Debbie R; van Adrichem, Marloes; Prakasa, Andreas D; Lozano-Torres, Jose L; Bakker, Jaap; Smant, Geert; Schots, Arjen

    2016-08-01

    Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing. PMID:26834022

  13. Comparison of Modules of Wild Type and Mutant Huntingtin and TP53 Protein Interaction Networks: Implications in Biological Processes and Functions

    Science.gov (United States)

    Basu, Mahashweta; Bhattacharyya, Nitai P.; Mohanty, Pradeep K.

    2013-01-01

    Disease-causing mutations usually change the interacting partners of mutant proteins. In this article, we propose that the biological consequences of mutation are directly related to the alteration of corresponding protein protein interaction networks (PPIN). Mutation of Huntingtin (HTT) which causes Huntington's disease (HD) and mutations to TP53 which is associated with different cancers are studied as two example cases. We construct the PPIN of wild type and mutant proteins separately and identify the structural modules of each of the networks. The functional role of these modules are then assessed by Gene Ontology (GO) enrichment analysis for biological processes (BPs). We find that a large number of significantly enriched () GO terms in mutant PPIN were absent in the wild type PPIN indicating the gain of BPs due to mutation. Similarly some of the GO terms enriched in wild type PPIN cease to exist in the modules of mutant PPIN, representing the loss. GO terms common in modules of mutant and wild type networks indicate both loss and gain of BPs. We further assign relevant biological function(s) to each module by classifying the enriched GO terms associated with it. It turns out that most of these biological functions in HTT networks are already known to be altered in HD and those of TP53 networks are altered in cancers. We argue that gain of BPs, and the corresponding biological functions, are due to new interacting partners acquired by mutant proteins. The methodology we adopt here could be applied to genetic diseases where mutations alter the ability of the protein to interact with other proteins. PMID:23741403

  14. Molecular basis of processing-induced changes in protein structure in relation to intestinal digestion in yellow and green type pea (Pisum sativum L.): A molecular spectroscopic analysis.

    Science.gov (United States)

    Yu, Gloria Qingyu; Warkentin, Tom; Niu, Zhiyuan; Khan, Nazir A; Yu, Peiqiang

    2015-12-01

    The objectives of this study were (1) to quantify the protein inherent molecular structural features of green cotyledon (CDC Striker) and yellow cotyledon (CDC Meadow) pea (Pisum sativum L.) seeds using molecular spectroscopic technique (FT/IR-ATR); (2) measure the denaturation of protein molecular makeup in the two types of pea during dry roasting (120°C for 60 min), autoclaving (120°C for 60 min) or microwaving (for 5 min); and (3) correlate the heat-induced changes in protein molecular makeup to the corresponding changes in protein digestibility determined using modified three-step in vitro procedure. Compared with yellow-type, the green-type peas had higher (Pyellow-type, the green-type peas had lower (Pyellow (r=0.81) pea-types. However, across the pea types the correlation was not significant. Principal component and hierarchical cluster analyses on the entire spectral data from the amide region (ca. 1727-1480 cm(-1)) were able to visualize and discriminate the structural difference between pea varieties and processing treatments. This study shows that the molecular spectroscopy can be used as a rapid tool to screen the protein value of raw and heat-treated peas. PMID:26188704

  15. Studies on the Coordination of Ribosomal Protein Assembly Events Involved in Processing and Stabilization of Yeast Early Large Ribosomal Subunit Precursors.

    Directory of Open Access Journals (Sweden)

    Uli Ohmayer

    Full Text Available Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins and ribosomal RNAs (rRNAs. Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs.

  16. Quantification of the main digestive processes in ruminants: the equations involved in the renewed energy and protein feed evaluation systems.

    Science.gov (United States)

    Sauvant, D; Nozière, P

    2016-05-01

    The evolution of feeding systems for ruminants towards evaluation of diets in terms of multiple responses requires the updating of the calculation of nutrient supply to the animals to make it more accurate on aggregated units (feed unit, or UF, for energy and protein digestible in the intestine, or PDI, for metabolizable protein) and to allow prediction of absorbed nutrients. The present update of the French system is based on the building and interpretation through meta-analysis of large databases on digestion and nutrition of ruminants. Equations involved in the calculation of UF and PDI have been updated, allowing: (1) prediction of the out flow rate of particles and liquid depending on the level of intake and the proportion of concentrate, and the use of this in the calculation of ruminal digestion of protein and starch from in situ data; (2) the system to take into account the effects of the main factors of digestive interactions (level of intake, proportion of concentrate, rumen protein balance) on organic matter digestibility, energy losses in methane and in urine; (3) more accurate calculation of the energy available in the rumen and the efficiency of its use for the microbial protein synthesis. In this renewed model UF and PDI values of feedstuffs vary depending on diet composition, and intake level. Consequently, standard feed table values can be considered as being only indicative. It is thus possible to predict the nutrient supply on a wider range of diets more accurately and in particular to better integrate energy×protein interactions occurring in the gut. PMID:26696120

  17. Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice

    OpenAIRE

    Hata, Kenji; Nishimura, Riko; Muramatsu, Shuji; Matsuda, Akio; Matsubara, Takuma; Amano, Katsuhiko; Ikeda, Fumiyo; Harley, Vincent R.; Yoneda, Toshiyuki

    2008-01-01

    The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an ess...

  18. Utilization of fisheries by-catch and processing wastes for lactic acid fermented silage and evaluation of degree of protein hydrolysis and in vitro digestibility

    OpenAIRE

    J. C. Ramírez- Ramírez; Huerta, S; Arias, L.; Prado, A.; Shirai, K.

    2008-01-01

    The purpose of this study was to produce protein hydrolysates from lactic acid fermentation of three sources of fish wastes: Shrimp by catch (SC), Sphyraena ensis wastes (SB) and mixture of fisheries processing wastes from several species (MixW). MixW were added with several sugar cane molasses concentrations as the carbon source, 180 g.kg-1 of sugar molasses gave the fastest acidification. The maximum concentration of lactic acid (Pmax) was significantly higher with Lactobacillus sp. B2 than...

  19. Effect of proteins on the surface microstructure evolution of a CoCrMo alloy in bio-tribocorrosion processes.

    Science.gov (United States)

    Wang, Zhongwei; Yan, Yu; Su, Yanjing; Qiao, Lijie

    2016-09-01

    Under tribological contact, the subsurface microstructure of CoCrMo alloys for artificial joint implants can be changed and affect the life and safety of such devices. As one of the most important and abundant components in the synovial fluid, proteins play a key role in affecting the bio-tribocorrosion behaviors of metal implants. The effect of proteins on the subsurface microstructure evolution of a CoCrMo alloy was investigated using a transmission electron microscope (TEM) in this study. The result shows that proteins have two main effects on the subsurface's evolution: forming a multilayered structure and causing severer subsurface deformation. The tribo-film can protect the passive film from scrapping, and then the passive film can reduce or even suppress the stacking fault annihilation by blocking the access to the metal surface. It leads to the stacking fault being diffused towards the deeper area and a strain accumulation in the subsurface, before inducing a severer deformation. On the other hand, the effect of proteins results in the location changing from the top surface to be underneath the top surface, where the maximum frictional shear stress occurs. This can cause a deeper deformation. PMID:27182652

  20. Behavior of Escherichia coli bacteria in whey protein and corn meal during twin screw extrusion processing at different temperatures

    Science.gov (United States)

    Many studies on the development of new and/ or value added nutritional meal corn and whey protein isolates for US consumers have been reported. However, information on the effect of treatment parameters on microbial safety of foods extruded below 100 deg C is limited. In this study, we investigated ...

  1. Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

    DEFF Research Database (Denmark)

    Ferreira, Gb; Overbergh, L; Hansen, Kasper Lage; D'Hertog, W; Hansen, Daniel Aaen; Maris, M; Moreau, Y; Workman, Christopher; Waelkens, E; Mathieu, C

    2008-01-01

    Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expressi...

  2. Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Gullberg, Maria; Li, Jiong;

    2013-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3Cpro) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3Cpro can be expected to be produced at equivalent concentrations. However, using...... with that achieved with a single P1-2A-3C polyprotein. Expression of the FMDV 3Cpro is poorly tolerated by mammalian cells and higher levels of the 3Cpro greatly inhibit protein expression. In addition, it is demonstrated that both the intact P1-2A precursor and the processed capsid proteins can be efficiently...... detected by FMDV antigen detection assays. Furthermore, the P1-2A and the processed forms each bind to the integrin αvβ6, the major FMDV receptor. These results contribute to the development of systems which efficiently express the components of empty capsid particles and may represent the basis for safer...

  3. Amino acid profile of raw and locally processed seeds of Prosopis africana and Ricinus communis: potential antidotes to protein malnutrition

    Directory of Open Access Journals (Sweden)

    Chidi U. Igwe

    2012-04-01

    Full Text Available Background: Increasing incidence of malnutrition occasioned by high incidence of hunger,worsening food situation in the world, insufficient availability and high cost of animal protein sources, has necessitated extensive research into and use of alternative plant protein sources especially underexploited leguminous seeds.Methods: Flours from raw, boiled and fermented seeds of Prosopis africana and Ricinus communis were evaluated for crude protein and amino acid (AA profiles, and their protein qualities determined. Results: Fermentation improved the protein contents of raw seeds of P. africana and R. communis by 18.70% and 3.95% respectively. In the raw and fermented P. africana seeds, glutamate at 132.60 ± 1.30 and 182.70 ± 3.02 mg/g crude protein (mg/gcp was the most abundant amino acid (AA, while leucine (62.80 ± 0.60 and 79.50 ± 2.01 mg/gcp was the most concentrated essential amino acid (EAA. Aspartate (151.90 ± 2.01 and 170.10 ± 2.00 mg/gcp and arginine (72.80 ± 2.01 and 78.60 ± 2.00 mg/gcp were the most concentrated and abundant non-essential amino acid (NEAA and EAA in the raw and fermented samples of R. communisrespectively. The total AA concentrations (mg/gcp of raw and fermented P. africana were 733.00 and 962.60 respectively, while those of R. communis were 823.50 and 894.10 respectively. The total EAA contents (mg/gcp for P. africana were 311.00 (raw and 404.50 (fermented, and for R. communis; 401.10 (raw and 430.30 (fermented. Threonine was the limiting EAA in raw and fermented P. africana, whereas lysine was the limiting EAA in R. communis raw sample. Fermentation significantly (p<0.05 increased the individual AA compositions of P. africana and R. communis by 94% and 53% respectively, while boiling reduced these parameters significantly (p<0.05 by 47% and 82% respectively. Conclusion: P. africana and R. communis seeds are potentially important plant sources of protein and essential amino acids, and so could be of great

  4. Development of Electronic Nose and Near Infrared Spectroscopy Analysis Techniques to Monitor the Critical Time in SSF Process of Feed Protein

    Directory of Open Access Journals (Sweden)

    Hui Jiang

    2014-10-01

    Full Text Available In order to assure the consistency of the final product quality, a fast and effective process monitoring is a growing need in solid state fermentation (SSF industry. This work investigated the potential of non-invasive techniques combined with the chemometrics method, to monitor time-related changes that occur during SSF process of feed protein. Four fermentation trials conducted were monitored by an electronic nose device and a near infrared spectroscopy (NIRS spectrometer. Firstly, principal component analysis (PCA and independent component analysis (ICA were respectively applied to the feature extraction and information fusion. Then, the BP_AdaBoost algorithm was used to develop the fused model for monitoring of the critical time in SSF process of feed protein. Experimental results showed that the identified results of the fusion model are much better than those of the single technique model both in the training and validation sets, and the complexity of the fusion model was also less than that of the single technique model. The overall results demonstrate that it has a high potential in online monitoring of the critical moment in SSF process by use of integrating electronic nose and NIRS techniques, and data fusion from multi-technique could significantly improve the monitoring performance of SSF process.

  5. Protein folding, protein homeostasis, and cancer

    Institute of Scientific and Technical Information of China (English)

    John H. Van Drie

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

  6. The tryptophan-rich sensory protein (TSPO is involved in stress-related and light-dependent processes in the cyanobacterium Fremyella diplosiphon

    Directory of Open Access Journals (Sweden)

    Andrea eBusch

    2015-12-01

    Full Text Available The tryptophan-rich sensory protein (TSPO is a membrane protein, which is a member of the 18 kilodalton translocator protein/peripheral-type benzodiazepine receptor (MBR family of proteins that is present in most organisms and is also referred to as Translocator protein 18 kDa. Although TSPO is associated with stress- and disease-related processes in organisms from bacteria to mammals, full elucidation of the functional role of the TSPO protein is lacking for most organisms in which it is found. In this study, we describe the regulation and function of a TSPO homolog in the cyanobacterium Fremyella diplosiphon, designated FdTSPO. Accumulation of the FdTSPO transcript is upregulated by green light and in response to nutrient deficiency and stress. A F. diplosiphon TSPO deletion mutant (i.e., ΔFdTSPO showed altered responses compared to the wild type strain under stress conditions, including salt treatment, osmotic stress and induced oxidative stress. Under salt stress, the FdTSPO transcript is upregulated and a ΔFdTSPO mutant accumulates lower levels of reactive oxygen species (ROS and displays increased growth compared to WT. In response to osmotic stress, FdTSPO transcript levels are upregulated and ΔFdTSPO mutant cells exhibit impaired growth compared to the wild type. By comparison, methyl viologen-induced oxidative stress results in higher ROS levels in the ΔFdTSPO mutant compared to the wild type strain. Taken together, our results provide support for the involvement of membrane-localized FdTSPO in mediating cellular responses to stress in F. diplosiphon and represent detailed functional analysis of a cyanobacterial TSPO. This study advances our understanding of the functional roles of TSPO homologs in vivo.

  7. Millisecond Timescale Dynamics of Human Liver Fatty Acid Binding Protein: Testing of Its Relevance to the Ligand Entry Process

    OpenAIRE

    Long, Dong; Yang, Daiwen

    2010-01-01

    For over a decade, scientists have been attempting to know more about the conformational dynamics of fatty acid binding proteins (FABPs), to answer the puzzling question of how ligands could access the internalized binding site(s). Conformational exchange of FABPs on the microsecond to millisecond timescales has been found in many FABPs and offers an important hypothesis for the ligand entry mechanism. Despite the potential significance, the validity of this hypothesis has not been verified y...

  8. CRISPR-Cas systems in the cyanobacterium Synechocystis sp. PCC6803 exhibit distinct processing pathways involving at least two Cas6 and a Cmr2 protein.

    Directory of Open Access Journals (Sweden)

    Ingeborg Scholz

    Full Text Available The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats--CRISPR associated proteins system provides adaptive immunity in archaea and bacteria. A hallmark of CRISPR-Cas is the involvement of short crRNAs that guide associated proteins in the destruction of invading DNA or RNA. We present three fundamentally distinct processing pathways in the cyanobacterium Synechocystis sp. PCC6803 for a subtype I-D (CRISPR1, and two type III systems (CRISPR2 and CRISPR3, which are located together on the plasmid pSYSA. Using high-throughput transcriptome analyses and assays of transcript accumulation we found all CRISPR loci to be highly expressed, but the individual crRNAs had profoundly varying abundances despite single transcription start sites for each array. In a computational analysis, CRISPR3 spacers with stable secondary structures displayed a greater ratio of degradation products. These structures might interfere with the loading of the crRNAs into RNP complexes, explaining the varying abundancies. The maturation of CRISPR1 and CRISPR2 transcripts depends on at least two different Cas6 proteins. Mutation of gene sll7090, encoding a Cmr2 protein led to the disappearance of all CRISPR3-derived crRNAs, providing in vivo evidence for a function of Cmr2 in the maturation, regulation of expression, Cmr complex formation or stabilization of CRISPR3 transcripts. Finally, we optimized CRISPR repeat structure prediction and the results indicate that the spacer context can influence individual repeat structures.

  9. Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

    Directory of Open Access Journals (Sweden)

    Shan Lu

    Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

  10. MUC16/CA125 in the Context of Modular Proteins with an Annotated Role in Adhesion-Related Processes: In Silico Analysis

    Directory of Open Access Journals (Sweden)

    Ninoslav Mitic

    2012-08-01

    Full Text Available Mucin 16 (MUC16 is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is no detailed insight into their heterogeneity and ligand properties, which may greatly influence its function and biomarker potential. This study was aimed at obtaining further insight into the biological capacity of MUC16/CA125, using in silico analysis of corresponding mucin sequences, including similarity searches as well as GO (gene ontology-based function prediction. The results obtained pointed to the similarities within extracellular serine/threonine rich regions of MUC16 to sequences of proteins expressed in evolutionary distant taxa, all having in common an annotated role in adhesion-related processes. Specifically, a homology to conserved domains from the family of herpesvirus major outer envelope protein (BLLF1 was found. In addition, the possible involvement of MUC16/CA125 in carbohydrate-binding interactions or cellular transport of protein/ion was suggested.

  11. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

    Science.gov (United States)

    Senderskiy, Igor V; Timofeev, Sergey A; Seliverstova, Elena V; Pavlova, Olga A; Dolgikh, Viacheslav V

    2014-01-01

    Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs. PMID:24705470

  12. Secretion of Antonospora (Paranosema locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

    Directory of Open Access Journals (Sweden)

    Igor V Senderskiy

    Full Text Available Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species, strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

  13. Single Molecule Analysis of the Arabidopsis FRA1 Kinesin Shows that It Is a Functional Motor Protein with Unusually High Processivity

    Institute of Scientific and Technical Information of China (English)

    Chuanmei Zhu; Ram Dixit

    2011-01-01

    The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism.The cortical localization of this kinesin during interphase raises the possibility that it transports cell wallrelated cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning.To determine whether FRA1 is an authentic motor protein,we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant,GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)-GFP).We found that FRA1(707)-GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated by the presence of microtubules.Using single molecule studies,we found that FRA1(707)-GFP moves processively along microtubule tracks at a velocity of about 0.4 μm s-1.In addition,we found that FRA1(707)-GFP is a microtubule plus-end-directed motor and that it moves along microtubules as a dimer.Interestingly,our single molecule analysis shows that the processivity of FRA1(707)-GFP is at least twice the processivity of conventional kinesin,making FRA1 the most processive kinesin to date.Together,our data show that FRA1 is a bona fide motor protein that has the potential to drive long-distance transport of cargo along cortical microtubules.

  14. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    Science.gov (United States)

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-01

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  15. Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

    LENUS (Irish Health Repository)

    2012-02-01

    Clathrin-mediated endocytosis (CME) is the best-characterized mechanism governing cellular membrane and protein trafficking. In this hypothesis review, we integrate recent evidence implicating CME and related cellular trafficking mechanisms in the pathophysiology of psychotic disorders such as schizophrenia and bipolar disorder. The evidence includes proteomic and genomic findings implicating proteins and genes of the clathrin interactome. Additionally, several important candidate genes for schizophrenia, such as dysbindin, are involved in processes closely linked to CME and membrane trafficking. We discuss that key aspects of psychosis neuropathology such as synaptic dysfunction, white matter changes and aberrant neurodevelopment are all influenced by clathrin-dependent processes, and that other cellular trafficking mechanisms previously linked to psychoses interact with the clathrin interactome in important ways. Furthermore, many antipsychotic drugs have been shown to affect clathrin-interacting proteins. We propose that the targeted pharmacological manipulation of the clathrin interactome may offer fruitful opportunities for novel treatments of schizophrenia.Molecular Psychiatry advance online publication, 11 October 2011; doi:10.1038\\/mp.2011.123.

  16. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    Science.gov (United States)

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. PMID:26049080

  17. Prediction of individual milk proteins including free amino acids in bovine milk using mid-infrared spectroscopy and their correlations with milk processing characteristics.

    Science.gov (United States)

    McDermott, A; Visentin, G; De Marchi, M; Berry, D P; Fenelon, M A; O'Connor, P M; Kenny, O A; McParland, S

    2016-04-01

    -infrared spectroscopy predictions (0.95). Weaker correlations among FAA were observed than the correlations among the protein fractions. Pearson correlations between gold standard protein fractions and the milk processing characteristics of rennet coagulation time, curd firming time, curd firmness, heat coagulating time, pH, and casein micelle size were weak to moderate and ranged from -0.48 (protein and pH) to 0.50 (total casein and a30). Pearson correlations between gold standard FAA and these milk processing characteristics were also weak to moderate and ranged from -0.60 (Val and pH) to 0.49 (Val and K20). Results from this study indicate that mid-infrared spectroscopy has the potential to predict protein fractions and some FAA in milk at a population level. PMID:26830742

  18. Toxicity, activation process, and histopathological effect of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 on Tuta absoluta.

    Science.gov (United States)

    Sellami, Sameh; Cherif, Maroua; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jamoussi, Kaïs

    2015-02-01

    Tuta absoluta is a destructive moth of Solanaceae plants and especially tomatoes. Here, we considered the entomopathogenic activity of the Bacillus thuringiensis Vip3Aa16 protein heterologously produced by Escherichia coli against T. absoluta. Purified Vip3Aa16 showed lower lethal concentration 50 % against third instar larvae (Toxin/tomato leaf) (335 ± 17 ng/cm(2)) compared to that of B. thuringiensis kurstaki HD1 δ-endotoxins (955 ± 4 ng/cm(2)) (P absoluta larva midguts consisted on a microvillus damage and an epithelial cell rupture. PMID:25432339

  19. The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

    OpenAIRE

    Zarbock Ralf; Woischnik Markus; Sparr Christiane; Thurm Tobias; Kern Sunčana; Kaltenborn Eva; Hector Andreas; Hartl Dominik; Liebisch Gerhard; Schmitz Gerd; Griese Matthias

    2012-01-01

    Abstract Background Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We al...

  20. Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes*

    OpenAIRE

    Haque, Farhana; Mazzeo, Daniela; Patel, Jennifer T; Smallwood, Dawn T.; Ellis, Juliet A; Shanahan, Catherine M.; Shackleton, Sue

    2009-01-01

    The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have ov...

  1. Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

    OpenAIRE

    Chaudhuri, Murari; Parris, Deborah S.

    2002-01-01

    The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities o...

  2. Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins

    DEFF Research Database (Denmark)

    Sasaki, T; Göhring, W; Mann, K; Brakebusch, C; Yamada, Y; Fässler, R; Timpl, R

    2001-01-01

    Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4-6, that...... processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo....

  3. How to Make a Non-Antigenic Protein (Auto) Antigenic: Molecular Complementarity Alters Antigen Processing and Activates Adaptive-Innate Immunity Synergy.

    Science.gov (United States)

    Root-Bernstein, Robert

    2015-01-01

    Evidence is reviewed that complementary proteins and peptides form complexes with increased antigenicity and/or autoimmunogenicity. Five case studies are highlighted: 1) diphtheria toxin-antitoxin (antibody), which induces immunity to the normally non-antigenic toxin, and autoimmune neuritis; 2) tryptophan peptide of myelin basic protein and muramyl dipeptide ("adjuvant peptide"), which form a complex that induces experimental allergic encephalomyelitis; 3) an insulin and glucagon complex that is far more antigenic than either component individually; 4) various causes of experimental autoimmune myocarditis such as C protein in combination with its antibody, or coxsackie B virus in combination with the coxsackie and adenovirus receptor; 5) influenza A virus haemagglutinin with the outer membrane protein of the Haemophilus influenzae, which increases antigenicity. Several mechanisms cooperate to alter immunogenicity. Complexation alters antigen processing, protecting the components against proteolysis, altering fragmentation and presenting novel antigens to the immune system. Complementary antigens induce complementary adaptive immune responses (complementary antibodies and/or T cell receptors) that produce circulating immune complexes (CIC). CIC stimulate innate immunity. Concurrently, complementary antigens stimulate multiple Toll-like receptors that synergize to over-produce cytokines, which further stimulate adaptive immunity. Thus innate and adaptive immunity form a positive feedback loop. If components of the complex mimic a host protein, then autoimmunity may result. Enhanced antigenicity for production of improved vaccines and/or therapeutic autoimmunity (e.g., against cancer cells) might be achieved by using information from antibody or TCR recognition sites to complement an antigen; by panning for complements in randomized peptide libraries; or using antisense peptide strategies to design complements. PMID:26179268

  4. Production and characterization of poly(3-hydroxybutyrate) generated by Alcaligenes latus using lactose and whey after acid protein precipitation process.

    Science.gov (United States)

    Berwig, Karina Hammel; Baldasso, Camila; Dettmer, Aline

    2016-10-01

    Whey after acid protein precipitation was used as substrate for PHB production in orbital shaker using Alcaligenes latus. Statistical analysis determined the most appropriate hydroxide for pH neutralization of whey after protein precipitation among NH4OH, KOH and NaOH 10%w/v. The results were compared to those of commercial lactose. A scale-up test in a 4L bioreactor was done at 35°C, 750rpm, 7L/min air flow, and 6.5 pH. The PHB was characterized through Fourier Transform Infrared Spectroscopy, thermogravimetry and differential scanning calorimetry. NH4OH provided the best results for productivity (p), 0.11g/L.h, and for polymer yield, (YP/S), 1.08g/g. The bioreactor experiment resulted in lower p and YP/S. PHB showed maximum degradation temperature (291°C), melting temperature (169°C), and chemical properties similar to those of standard PHB. The use of whey as a substrate for PHB production did not affect significantly the final product quality. PMID:27347795

  5. LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages.

    Science.gov (United States)

    Ruggiero, Tina; Trabucchi, Michele; De Santa, Francesca; Zupo, Simona; Harfe, Brian D; McManus, Michael T; Rosenfeld, M Geoff; Briata, Paola; Gherzi, Roberto

    2009-09-01

    The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS. PMID:19423639

  6. Characterization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate Bayesian approach.

    Science.gov (United States)

    Fu, Zhibiao; Baker, Daniel; Cheng, Aili; Leighton, Julie; Appelbaum, Edward; Aon, Juan

    2016-05-01

    The principle of quality by design (QbD) has been widely applied to biopharmaceutical manufacturing processes. Process characterization is an essential step to implement the QbD concept to establish the design space and to define the proven acceptable ranges (PAR) for critical process parameters (CPPs). In this study, we present characterization of a Saccharomyces cerevisiae fermentation process using risk assessment analysis, statistical design of experiments (DoE), and the multivariate Bayesian predictive approach. The critical quality attributes (CQAs) and CPPs were identified with a risk assessment. The statistical model for each attribute was established using the results from the DoE study with consideration given to interactions between CPPs. Both the conventional overlapping contour plot and the multivariate Bayesian predictive approaches were used to establish the region of process operating conditions where all attributes met their specifications simultaneously. The quantitative Bayesian predictive approach was chosen to define the PARs for the CPPs, which apply to the manufacturing control strategy. Experience from the 10,000 L manufacturing scale process validation, including 64 continued process verification batches, indicates that the CPPs remain under a state of control and within the established PARs. The end product quality attributes were within their drug substance specifications. The probability generated with the Bayesian approach was also used as a tool to assess CPP deviations. This approach can be extended to develop other production process characterization and quantify a reliable operating region. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:799-812, 2016. PMID:27095416

  7. Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

    International Nuclear Information System (INIS)

    The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2α) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2α. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2α. Confocal fluorescence microscopy revealed that a subpopulation of AP2α was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin β and Nup153, implying that AP2α negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2α may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle

  8. A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells.

    Science.gov (United States)

    Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi

    2016-01-01

    Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression. PMID:26153462

  9. The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. Extraction of protein and application to immunoblotting.

    OpenAIRE

    Y. Sato; Mukai, K.; Furuya, S; Kameya, T.; Hirohashi, S

    1992-01-01

    The authors have previously reported a new fixation and paraffin-embedding method (the AMeX method), which preserves many antigens as well as high molecular-weight DNA and RNA that are normally destroyed by the routine formalin fixation and paraffin-embedding process. In the present study, the authors analyzed the preservation of protein suitable for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting in tissue fixed by the AMeX method. The method used for ...

  10. Mutations in the RNA Binding Domain of Stem-Loop Binding Protein Define Separable Requirements for RNA Binding and for Histone Pre-mRNA Processing

    OpenAIRE

    Dominski, Zbigniew; Erkmann, Judith A.; Greenland, John A.; Marzluff, William F

    2001-01-01

    Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3′ untranslated region of histone pre-mRNAs and facilitate 3′ end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial lo...

  11. Utilization of Solid Waste of Tofu and Out of Grade Carrot as a Source of Vegetable Protein and Fibre in Nugget Processing

    OpenAIRE

    Evawati Evawati; Irwan Roza

    2014-01-01

    Tofu residue production is a solid by-product of the tofu industry, which contains high protein and can be processed for food consumption. It has a good taste and is safe to consume as a chicken meat substitute in the form of nuggets they are called de-soya nuggets. One of the methods to decrease bad health effects from fast food is to use tofu nuggets made from the Tofu waste by fortification with local food ingredients that are rich in fiber and antioxidants, similar with out-of-grade carro...

  12. Nuclear LSm8 affects number of cytoplasmic processing bodies via controlling cellular distribution of Like-Sm proteins

    Czech Academy of Sciences Publication Activity Database

    Novotný, Ivan; Podolská, Kateřina; Blažíková, Michaela; Valášek, Leoš; Svoboda, Petr; Staněk, David

    2012-01-01

    Roč. 23, č. 19 (2012), s. 3776-3785. ISSN 1059-1524 R&D Projects: GA AV ČR KAN200520801; GA ČR GA204/07/0133; GA ČR GAP305/10/2215; GA ČR GAP302/11/1910; GA ČR(CZ) GBP305/12/G034 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50520514; CEZ:AV0Z50200510 Institutional support: RVO:68378050 ; RVO:68378041 ; RVO:61388971 Keywords : P-bodies * LSm proteins * mRNA degradation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.604, year: 2012

  13. Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Cowell, G M

    1986-01-01

    Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3...... microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression. The...

  14. Hidrolisado protéico de pescado obtido por vias química e enzimática a partir de corvina (Micropogonias furnieri) Fish protein hydrolyzed obtained by chemical and enzymatic processes from corvina (Micropogonias furnieri)

    OpenAIRE

    Vilásia Guimarães Martins; Jorge Alberto Vieira Costa; Carlos Prentice-Hernández

    2009-01-01

    The fish proteins has the advantage of a high sensibility to the hydrolysis and also a balanced composition in aminoacids. The production of protein hydrolyzed from by-products of fish process industry has been receiving more attention on the last years. The aim of this work was to evaluate the production of protein hydrolyzed from Micropogonias furnieri through chemical and enzymatic methods, verifying some functional properties. The results showed that the production of the hydrolyzed impro...

  15. Chemogenomic Study of Carboplatin in Saccharomyces cerevisiae: Inhibition of the NEDDylation Process Overcomes Cellular Resistance Mediated by HuR and Cullin Proteins

    Science.gov (United States)

    Custodio, Débora Fernandes; Freitas, Vanessa Morais; Monteiro, Gisele

    2015-01-01

    The use of carboplatin in cancer chemotherapy is limited by the emergence of drug resistance. To understand the molecular basis for this resistance, a chemogenomic screen was performed in 53 yeast mutants that had previously presented strong sensitivity to this widely used anticancer agent. Thirty-four mutants were responsive to carboplatin, and from these, 21 genes were selected for further studies because they have human homologues. Sixty percent of these yeast genes possessed human homologues which encoded proteins that interact with cullin scaffolds of ubiquitin ligases, or whose mRNA are under the regulation of Human antigen R (HuR) protein. Both HuR and cullin proteins are regulated through NEDDylation post-translational modification, and so our results indicate that inhibition of this process should sensitise resistant tumour cells to carboplatin. We showed that treatment of a tumour cell line with MLN4924, a NEDDylation inhibitor, overcame the resistance to carboplatin. Our data suggest that inhibition of NEDDylation may be a useful strategy to resensitise tumour cells in patients that have acquired carboplatin resistance. PMID:26692264

  16. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

    2012-10-10

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

  17. Effects of various feed supplements containing fish protein hydrolysate or fish processing by-products on the innate immune functions of juvenile coho salmon (oncorhynchus kisutch)

    Science.gov (United States)

    Murray, A.L.; Pascho, R.J.; Alcorn, S.W.; Fairgrieve, W.T.; Shearer, K.D.; Roley, D.

    2003-01-01

    Immunomodulators administered to fish in the diet have been shown in some cases to enhance innate immune defense mechanisms. Recent studies have suggested that polypeptide fractions found in fish protein hydrolysates may stimulate factors in fish important for disease resistance. For the current study, groups of coho salmon were reared on practical feeds that contained either fish meal (Control diet), fish meal supplemented with cooked fish by-products, or fish meal supplemented with hydrolyzed fish protein alone, or with hydrolyzed fish protein and processed fish bones. For each diet group, three replicate tanks of fish were fed the experimental diets for 6 weeks. Morphometric measurements, and serologic and cellular assays were used to evaluate the general health and immunocompetence of fish in the various feed groups. Whereas the experimental diets had no effect on the morphometric and cellular measurements, fish fed cooked by-products had increased leucocrit levels and lower hematocrit levels than fish from the other feed groups. Innate cellular responses were increased in all feed groups after feeding the four experimental diets compared with pre-feed results. Subgroups of fish from each diet group were also challenged with Vibrio anguillarum (ca. 7.71 ?? 105 bacteria ml-1) at 15??C by immersion. No differences were found in survival among the various feed groups.

  18. The Effect of Oral Intake of Low-Temperature-Processed Whey Protein Concentrate on Colitis and Gene Expression Profiles in Mice

    Directory of Open Access Journals (Sweden)

    Sharmila Jayatilake

    2014-06-01

    Full Text Available Inflammatory bowel disease (IBD is an autoimmune disease of unknown etiology and can lead to inflammation and cancer. Whey proteins contain many bioactive peptides with potential health benefits against IBD. We investigated the effect of low-temperature-processed whey protein concentrate (LWPC on the suppression of IBD by using a dextran sodium sulfate (DSS-induced colitis model in BALB/c mice. Oral intake of LWPC resulted in improved recovery of body weight in mice. Histological analysis showed that the epithelium cells of LWPC-treated mice were healthier and that lymphocyte infiltration was reduced. The increase in mucin due to the LWPC also reflected reduced inflammation in the colon. Transcriptome analysis of the colon by DNA microarrays revealed marked downregulation of genes related to immune responses in LWPC-fed mice. In particular, the expression of interferon gamma receptor 2 (Ifngr2 and guanylate-binding proteins (GBPs was increased by DSS treatment and decreased in LWPC-fed mice. These findings suggest that LWPCs suppress DSS-induced inflammation in the colon by suppressing the signaling of these cytokines. Our findings suggest that LWPCs would be an effective food resource for suppressing IBD symptoms.

  19. NMR assignment of intrinsically disordered self-processing module of the FrpC protein of Neisseria meningitidis

    Czech Academy of Sciences Publication Activity Database

    Kubáň, V.; Nováček, J.; Bumba, Ladislav; Žídek, L.

    2015-01-01

    Roč. 9, č. 2 (2015), s. 435-440. ISSN 1874-2718 R&D Projects: GA ČR(CZ) GAP207/11/0717 Institutional support: RVO:61388971 Keywords : FrpC * Self-processing module * Neisseria meningitidis Subject RIV: EE - Microbiology, Virology Impact factor: 0.760, year: 2014

  20. Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Norén, Ove;

    1983-01-01

    The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo...

  1. Influence of disease process and duration on acute phase proteins in serum and peritoneal fluid of horses with colic

    DEFF Research Database (Denmark)

    Pihl, Tina; Scheepers, E.; Sanz, M.; Goddard, A.; Page, P.; Toft, Nils; Andersen, Pia Haubro; Jacobsen, Stine

    2015-01-01

    objective of this study was to investigate the influence of demographics (age, sex, breed), disease process (sim-ple obstruction, strangulating obstruction, inflammatory), disease location, disease duration, hypovolemia, and admission hospi-tal on concentrations of APP, lactate and white blood cell counts...

  2. Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein.

    Directory of Open Access Journals (Sweden)

    Jiang-Li Tan

    Full Text Available BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP, which represents the first step in the production of amyloid β (Aβ peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer's disease (AD. The association between oxidative stress (OS and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10-40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS, as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.

  3. BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

    Science.gov (United States)

    Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

    2016-11-01

    BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. PMID:27436242

  4. Study on processing of mung bean protein powder by spray drying%喷雾干燥法制备绿豆蛋白粉工艺研究

    Institute of Scientific and Technical Information of China (English)

    李玉邯

    2014-01-01

    为了便于绿豆蛋白提取液的保存和运输,研究了通过喷雾干燥法将绿豆蛋白提取液制备成绿豆蛋白粉的工艺条件和最优参数。在单因素试验的基础上,以进风温度、出风温度、进料速率为影响因素,进行 L9(34)正交试验。实验结果表明,最佳工艺参数为:进风温度160℃,出风温度70℃,进料流量25 mL/min。该工艺条件所制绿豆蛋白粉色泽佳,具有绿豆特殊的清香味,且集粉率高,可达56%。为绿豆蛋白粉的工业化生产提供了相关的基础性参数。%In order to storage and transportation of mung bean protein extract,the technique and optimal parameters for spray drying process of the extract were investigated.The effects of spray drying process parameters such as inlet air temperature,outlet air temperature and feeding velocity on the physicochemi-cal properties of mung bean protein powder were explored by single factor and L9 (34 )orthogonal experi-ment.The experiment results showed that the optimization spray drying process as followed:the inlet air temperature was 160 ℃,outlet air temperature 70 ℃,feeding velocity 25 mL/min.The achieved dried mung bean protein power had the advantages of beautiful color,good taste,and the powder collection rate was 56%.The results are expected to provide some fundamental data for the industrial production of mung bean protein powder.

  5. Sleep, Plasticity and the Pathophysiology of Neurodevelopmental Disorders: The Potential Roles of Protein Synthesis and Other Cellular Processes

    Directory of Open Access Journals (Sweden)

    Dante Picchioni

    2014-03-01

    Full Text Available Sleep is important for neural plasticity, and plasticity underlies sleep-dependent memory consolidation. It is widely appreciated that protein synthesis plays an essential role in neural plasticity. Studies of sleep-dependent memory and sleep-dependent plasticity have begun to examine alterations in these functions in populations with neurological and psychiatric disorders. Such an approach acknowledges that disordered sleep may have functional consequences during wakefulness. Although neurodevelopmental disorders are not considered to be sleep disorders per se, recent data has revealed that sleep abnormalities are among the most prevalent and common symptoms and may contribute to the progression of these disorders. The main goal of this review is to highlight the role of disordered sleep in the pathology of neurodevelopmental disorders and to examine some potential mechanisms by which sleep-dependent plasticity may be altered. We will also briefly attempt to extend the same logic to the other end of the developmental spectrum and describe a potential role of disordered sleep in the pathology of neurodegenerative diseases. We conclude by discussing ongoing studies that might provide a more integrative approach to the study of sleep, plasticity, and neurodevelopmental disorders.

  6. Transcranial laser therapy alters amyloid precursor protein processing and improves mitochondrial function in a mouse model of Alzheimer's disease

    Science.gov (United States)

    McCarthy, Thomas; Yu, Jin; El-Amouri, Salim; Gattoni-Celli, Sebastiano; Richieri, Steve; De Taboada, Luis; Streeter, Jackson; Kindy, Mark S.

    2011-03-01

    Transcranial laser therapy (TLT) using a near-infrared energy laser system was tested in the 2x Tg amyloid precursor protein (APP) mouse model of Alzheimer's Disease (AD). TLT was administered 3 times/week at escalating doses, starting at 3 months of age, and was compared to a control group which received no laser treatment. Treatment sessions were continued for a total of six months. The brains were examined for amyloid plaque burden, Aβ peptides (Aβ1-40 and Aβ1-42 ), APP cleavage products (sAPPα, CTFβ) and mitochondrial activity. Administration of TLT was associated with a significant, dose-dependent reduction in amyloid load as indicated by the numbers of Aβ plaques. Levels of Aβ1-40 and Aβ1-42 levels were likewise reduced in a dose-dependent fashion. All TLT doses produced an increase in brain sAPPα and a decrease in CTFβ levels consistent with an increase in α-secretase activity and a decrease in β-secretase activity. In addition, TLT increased ATP levels and oxygen utilization in treated animals suggesting improved mitochondrial function. These studies suggest that TLT is a potential candidate for treatment of AD.

  7. Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

    Science.gov (United States)

    Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

    2016-02-12

    Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

  8. Development a scalable production process for truncated human papillomavirus type-6 L1 protein using WAVE Bioreactor and hollow fiber membrane.

    Science.gov (United States)

    Sun, Bo; Zhao, Dandan; Zhang, Xizhen; Gu, Tiejun; Yu, XiangHui; Sun, Shiyang; Zhao, Xinghong; Wei, Liu; Liu, Dawei; Yan, Hui; Meng, Xiangyu; Kong, Wei; Xu, Fei; Yang, Ping; Jiang, Chunlai

    2016-02-01

    Here, we describe a process for expression, purification, and characterization of truncated human papillomavirus type-6 (HPV-6) L1 virus-like particles (VLPs). The scalable cultivation process in a WAVE Bioreactor at the 10-L scale was optimized to express HPV-6 L1 VLPs using the baculovirus insect expression system. A hollow fiber membrane system was used for the integrated operation, including concentration, diafiltration, extraction, and clarification. The HPV-6 L1 protein was further purified by anion-exchange chromatography and hydrophobic chromatography. The HPV-6 L1 protein could self-assemble into VLPs with a diameter of approximately 50-60 nm after removal of the reductant dithiothreitol (DTT). The final purified HPV-6 L1 VLPs product was characterized to estimate yield and purity, and exceeds the requirements for pharmaceutical-grade VLP vaccine. Immunization of mice demonstrated that the vaccine could elicit high titer neutralizing antibodies in vivo. This study confirms the feasibility of producing pharmaceutical-grade HPV type-6 L1 VLPs on an industrial scale for clinical trials. PMID:26446387

  9. Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

    Directory of Open Access Journals (Sweden)

    Vadim Shchepachev

    2012-10-01

    Full Text Available Clericuzio-type poikiloderma with neutropenia (PN is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

  10. Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process.

    Science.gov (United States)

    Welderufael, Fisseha Tesfay; Gibson, Trevor; Methven, Lisa; Jauregi, Paula

    2012-10-15

    The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC(50)=287 μg/mL) and (ii) from β-lactoglobulin (IC(50)=128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC(50)). PMID:23442643

  11. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused to the...... protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N-terminal of...... the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is...

  12. Cogeneration of hydrogen and methane from protein-mixed food waste by two-phase anaerobic process

    Energy Technology Data Exchange (ETDEWEB)

    Song, Wenlu; Cheng, Jun; Zhou, Junhu; Xie, Binfei; Su, Huibo; Cen, Kefa [State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027 (China)

    2010-04-15

    The cogeneration of hydrogen and methane from protein-mixed food waste by two-phase anaerobic fermentation was investigated for the first time in this paper. The hydrogen-producing bacteria derived from activated sludge were used to produce hydrogen from defatted milk powder (DMP) in the first stage, and Saccharomyces cerevisiae was used to promote the hydrogen production. The hydrogen yield from DMP with S. cerevisiae is promoted from 171.9 ml/g-TVS to 186.1 ml/g-TVS, while the peak hydrogen rate is promoted from 47.9 ml/h to 81.3 ml/h. The residual solutions from the first H{sub 2}-producing stage were reutilized by methanogen community to further produce methane in the second stage. Over 96 wt % of acetic acid and butyric acid in the residual solution from DMP with S. cerevisiae are reutilized to give a methane yield of 209.7 ml/g-TVS and peak methane rate of 411.7 ml/d. The cogeneration of hydrogen and methane from DMP markedly increases the energy conversion efficiency from 10.85-11.75% in only hydrogen production to 55.58-61.96%. Because lactose in DMP cogenerates hydrogen yield of 236.5 ml/g-TVS and methane yield of 263.7 ml/g-TVS, it is concluded that lactoprotein in DMP cogenerates hydrogen yield of 136.5 ml/g-TVS and methane yield of 157.8 ml/g-TVS by two-phase fermentation. (author)

  13. Assessment of the Sensitizing Potential of Processed Peanut Proteins in Brown Norway Rats: Roasting Does Not Enhance Allergenicity

    DEFF Research Database (Denmark)

    Kroghsbo, Stine; Rigby, Neil M.; Johnson, Philip L F;

    2014-01-01

    intraperitoneal route. Methods Sensitization potential of processed peanut products and Ara h 1 was examined in Brown Norway (BN) rats by oral administration of blanched or oil-roasted peanuts or peanut butter or by intraperitoneal immunization of purified native (N-), heated (H-) or heat glycated (G-)Ara h 1....... Levels of specific IgG and IgE were determined by ELISA and IgE functionality was examined by rat basophilic leukemia (RBL) cell assay. Results In rats dosed orally, roasted peanuts induced significant higher levels of specific IgE to NAra h 1 and 2 than blanched peanuts or peanut butter but with the...

  14. Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci.

    Science.gov (United States)

    Alfieri, Beatrice; Folloni, Silvia; Elviri, Lisa; Gobbo, Marina; Berni, Rodolfo; Folli, Claudia

    2008-08-01

    The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4mg/l of cell culture for TraC and up to 10mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active. PMID:18468916

  15. Protein engineering of a bacterial N-acyl-d-glucosamine 2-epimerase for improved stability under process conditions.

    Science.gov (United States)

    Klermund, Ludwig; Riederer, Amelie; Hunger, Annique; Castiglione, Kathrin

    2016-06-01

    Enzymatic cascade reactions, i.e. the combination of several enzyme reactions in one pot without isolation of intermediates, have great potential for the establishment of sustainable chemical processes. However, many cascade reactions suffer from cross-inhibitions and enzyme inactivation by components of the reaction system. This study focuses on the two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-d-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE) in combination with an N-acetylneuraminate lyase (NAL) from Escherichia coli. AvaAGE epimerizes N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), which then reacts with pyruvate in a NAL-catalyzed aldol condensation to form Neu5Ac. However, AvaAGE is inactivated by high pyruvate concentrations, which are used to push the NAL reaction toward the product side. A biphasic inactivation was observed in the presence of 50-800mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis revealed that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE. Because ATP is an allosteric activator of the epimerase and the binding of the nucleotide is crucial for its catalytic properties, saturation mutagenesis at position K160 was performed to identify the most compatible amino acid exchanges. The best variants, K160I, K160N and K160L, showed no inactivation by pyruvate, but significantly impaired kinetic parameters. For example, depending on the mutant, the turnover number kcat was reduced by 51-68% compared with the wild-type enzyme. A mechanistic model of the Neu5Ac synthesis was established, which can be used to select the AvaAGE variant that is most favorable for a given process condition. The results show that mechanistic models can greatly facilitate the choice of the right enzyme for an enzymatic cascade reaction with multiple cross-inhibitions and inactivation phenomena

  16. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    International Nuclear Information System (INIS)

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: → A two-hybrid assay was developed to study interactions in macromolecular complexes. → The assay was applied to interactions between components of mRNA P-bodies. → The assay effectively and efficiently identified protein interaction domains. → P-body assembly in mammalian cells differs from that in other species.

  17. Baicalein reduces β-amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer’s disease transgenic mouse model

    Science.gov (United States)

    Zhang, She-Qing; Obregon, Demian; Ehrhart, Jared; Deng, Juan; Tian, Jun; Hou, Huayan; Giunta, Brian; Sawmiller, Darrell; Tan, Jun

    2013-01-01

    Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ-aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABAA modulation for Alzheimer’s disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild-type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β-amyloid (Aβ) by increasing APP α-processing. These effects were blocked by the GABAA antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α-secretase processing, reduced Aβ production, and reduced AD-like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABAA receptors. © 2013 Wiley Periodicals, Inc. PMID:23686791

  18. The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

    Directory of Open Access Journals (Sweden)

    Ofir Bahar

    2014-01-01

    Full Text Available Pattern recognition receptors (PRRs play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo. In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

  19. Utilization of Solid Waste of Tofu and Out of Grade Carrot as a Source of Vegetable Protein and Fibre in Nugget Processing

    Directory of Open Access Journals (Sweden)

    Evawati Evawati

    2014-01-01

    Full Text Available Tofu residue production is a solid by-product of the tofu industry, which contains high protein and can be processed for food consumption. It has a good taste and is safe to consume as a chicken meat substitute in the form of nuggets they are called de-soya nuggets. One of the methods to decrease bad health effects from fast food is to use tofu nuggets made from the Tofu waste by fortification with local food ingredients that are rich in fiber and antioxidants, similar with out-of-grade carrot. From the research it can be concluded that the substitution of tofu waste and carrot fortifications as de-soya nuggets can increase the nutritional value and fiber of food. Nuggets known as fast foods have high amounts of fat and cholesterols because they are made from animal fat and have low amount fiber. The number of nutritional values that fulfill the standard for a de-soya nugget is, with the largest substitute being of 30% tofu waste with carrot fortifications of 15% that contains 12.53% protein, 30.1% carbohydrate,  11,83% fat, 42.8% water, 1,8700% ash, 8,05% dietary fiber , and  less than 3.0 x 103 (1.8 x 103 colonies/gr total microbial.

  20. Chemical composition and molecular structure of polysaccharide-protein biopolymer from Durio zibethinus seed: extraction and purification process

    Directory of Open Access Journals (Sweden)

    Amid Bahareh

    2012-10-01

    Full Text Available Abstract Background The biological functions of natural biopolymers from plant sources depend on their chemical composition and molecular structure. In addition, the extraction and further processing conditions significantly influence the chemical and molecular structure of the plant biopolymer. The main objective of the present study was to characterize the chemical and molecular structure of a natural biopolymer from Durio zibethinus seed. A size-exclusion chromatography coupled to multi angle laser light-scattering (SEC-MALS was applied to analyze the molecular weight (Mw, number average molecular weight (Mn, and polydispersity index (Mw/Mn. Results The most abundant monosaccharide in the carbohydrate composition of durian seed gum were galactose (48.6-59.9%, glucose (37.1-45.1%, arabinose (0.58-3.41%, and xylose (0.3-3.21%. The predominant fatty acid of the lipid fraction from the durian seed gum were palmitic acid (C16:0, palmitoleic acid (C16:1, stearic acid (C18:0, oleic acid (C18:1, linoleic acid (C18:2, and linolenic acid (C18:2. The most abundant amino acids of durian seed gum were: leucine (30.9-37.3%, lysine (6.04-8.36%, aspartic acid (6.10-7.19%, glycine (6.07-7.42%, alanine (5.24-6.14%, glutamic acid (5.57-7.09%, valine (4.5-5.50%, proline (3.87-4.81%, serine (4.39-5.18%, threonine (3.44-6.50%, isoleucine (3.30-4.07%, and phenylalanine (3.11-9.04%. Conclusion The presence of essential amino acids in the chemical structure of durian seed gum reinforces its nutritional value.

  1. Arabinogalactan proteins

    DEFF Research Database (Denmark)

    Knoch, Eva; Dilokpimol, Adiphol; Geshi, Naomi

    2014-01-01

    Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant...

  2. A computational approach identifies two regions of Hepatitis C Virus E1 protein as interacting domains involved in viral fusion process

    Directory of Open Access Journals (Sweden)

    El Sawaf Gamal

    2009-07-01

    Full Text Available Abstract Background The E1 protein of Hepatitis C Virus (HCV can be dissected into two distinct hydrophobic regions: a central domain containing an hypothetical fusion peptide (FP, and a C-terminal domain (CT comprising two segments, a pre-anchor and a trans-membrane (TM region. In the currently accepted model of the viral fusion process, the FP and the TM regions are considered to be closely juxtaposed in the post-fusion structure and their physical interaction cannot be excluded. In the present study, we took advantage of the natural sequence variability present among HCV strains to test, by purely sequence-based computational tools, the hypothesis that in this virus the fusion process involves the physical interaction of the FP and CT regions of E1. Results Two computational approaches were applied. The first one is based on the co-evolution paradigm of interacting peptides and consequently on the correlation between the distance matrices generated by the sequence alignment method applied to FP and CT primary structures, respectively. In spite of the relatively low random genetic drift between genotypes, co-evolution analysis of sequences from five HCV genotypes revealed a greater correlation between the FP and CT domains than respect to a control HCV sequence from Core protein, so giving a clear, albeit still inconclusive, support to the physical interaction hypothesis. The second approach relies upon a non-linear signal analysis method widely used in protein science called Recurrence Quantification Analysis (RQA. This method allows for a direct comparison of domains for the presence of common hydrophobicity patterns, on which the physical interaction is based upon. RQA greatly strengthened the reliability of the hypothesis by the scoring of a lot of cross-recurrences between FP and CT peptides hydrophobicity patterning largely outnumbering chance expectations and pointing to putative interaction sites. Intriguingly, mutations in the CT

  3. Serum protein removal from skim milk with a 3-stage, 3× ceramic Isoflux membrane process at 50°C.

    Science.gov (United States)

    Adams, Michael C; Barbano, David M

    2013-04-01

    Small pore microfiltration (MF) can be used to remove serum proteins (SP) from skim milk. The process's SP removal efficiency directly influences the technology's economic feasibility. Our objective was to quantify the capacity of 0.14μm ceramic Isoflux MF membranes (TAMI, Nyons, France) to remove SP from skim milk. A 3-stage, 3×, feed-and-bleed MF study with diafiltration in the latter 2 stages was conducted at 50°C using Isoflux membranes to determine cumulative SP removal percentages and SP removal rates at each processing stage. The experiment was replicated 3 times starting with 3 separate lots of raw milk. In contrast to 3× MF theoretical cumulative SP removal percentages of 68, 90, and 97% after 1, 2, and 3 stages, respectively, the 3× Isoflux MF process removed only 39.5, 58.4, and 70.2% of SP after 1, 2, and 3 stages, respectively. Previous research has been published that provides the skim milk SP removal capacities of 3-stage, 3× 0.1μm ceramic Membralox (Pall Corp., Cortland, NY) uniform transmembrane pressure (UTP), 0.1μm ceramic Membralox graded permeability (GP), and 0.3μm polymeric polyvinylidene fluoride spiral-wound (PVDF-SW) MF systems (Parker-Hannifin, Process Advanced Filtration Division, Tell City, IN) at 50°C. No difference in cumulative SP removal percentage after 3 stages was detected between the Isoflux and previously published PVDF-SW values (70.3%), but SP removal was lower than published GP (96.5%) and UTP (98.3%) values. To remove 95% of SP from 1,000kg of skim milk in 12h it would take 7, 3, 3, and 7 stages with 6.86, 1.91, 2.82, and 17.98m(2) of membrane surface area for the Isoflux, GP, UTP, and PVDF-SW systems, respectively. The MF systems requiring more stages would produce additional permeate at lower protein concentrations. The ceramic MF systems requiring more surface area would incur higher capital costs. The authors hypothesize that SP removal with the Isoflux membranes was lower than theoretical for the following

  4. New approach for predicting protein-protein interactions

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Protein-protein interactions (PPIs) are of vital importance for virtually all processes of a living cell. The study of these associations of protein molecules could improve people's understanding of diseases and provide basis for therapeutic approaches.

  5. 灵芝水溶性蛋白提取工艺优化%Extraction process optimization of water-soluble protein from Ganoderma lucidum

    Institute of Scientific and Technical Information of China (English)

    姜沅彤; 赵岩; 蔡恩博; 刘双利; 杨鹤; 张连学; 王士杰

    2014-01-01

    Objective To optimize the extraction process of water-soluble protein from shell-broken spore powder and sporophore of Ganoderma lucidum. Methods The effects of extraction temperature, extraction time, the liquid to solid ratio and their interaction on water-soluble protein extraction rate were studied by central composite design. The predictive model and reliability were developed by SAS software and response surface analysis, and the optimum extraction conditions were gotten by ridge analysis. Results The optimum extraction conditions of water-soluble protein from shell-broken spore powder and sporophore of Ganoderma lucidum as follows:extraction temperature 92℃, extraction time 196 min, the liquid to solid ratio 1:173. The optimum extraction conditions of water-soluble protein from sporophore of Ganoderma lucidum as follows:extraction temperature 94℃, extraction time 157 min, the liquid to solid ratio 1:166. Conclusion Under the optimum conditions, the extraction rate of water-soluble protein from shell-broken spore powder was 1.57%, and the extraction rate of the water-soluble protein from sporophore was 0.96%.%目的:优化破壁灵芝孢子粉及灵芝子实体中水溶性蛋白的提取工艺。方法采用中心组合设计方法,以提取温度、提取时间、固液比为考察因素,研究各因素及其交互作用对水溶性蛋白提取率的影响。应用SAS软件和响应面分析相结合的方法,模拟得到回归方程的预测模型和可信度,并通过岭脊分析得到最佳提取条件。结果破壁灵芝孢子粉中水溶性蛋白最佳的提取条件为:提取温度92℃、提取时间196 min、固液比1:173。而灵芝子实体的最佳提取工艺为:提取温度94℃、提取时间157 min、固液比1:166。结论最佳条件下孢子粉中水溶性蛋白的提取率为1.57%,而子实体中水溶性蛋白的提取率为0.96%。

  6. Proteomic Analysis of a Poplar Cell Suspension Culture Suggests a Major Role of Protein S-Acylation in Diverse Cellular Processes.

    Science.gov (United States)

    Srivastava, Vaibhav; Weber, Joseph R; Malm, Erik; Fouke, Bruce W; Bulone, Vincent

    2016-01-01

    S-acylation is a reversible post-translational modification of proteins known to be involved in membrane targeting, subcellular trafficking, and the determination of a great variety of functional properties of proteins. The aim of this work was to identify S-acylated proteins in poplar. The use of an acyl-biotin exchange method and mass spectrometry allowed the identification of around 450 S-acylated proteins, which were subdivided into three major groups of proteins involved in transport, signal transduction, and response to stress, respectively. The largest group of S-acylated proteins was the protein kinase superfamily. Soluble N-ethylmaleimide-sensitive factor-activating protein receptors, band 7 family proteins and tetraspanins, all primarily related to intracellular trafficking, were also identified. In addition, cell wall related proteins, including cellulose synthases and other glucan synthases, were found to be S-acylated. Twenty four of the identified S-acylated proteins were also enriched in detergent-resistant membrane microdomains, suggesting S-acylation plays a key role in the localization of proteins to specialized plasma membrane subdomains. This dataset promises to enhance our current understanding of the various functions of S-acylated proteins in plants. PMID:27148305

  7. A biodegradable polymeric system for peptide–protein delivery assembled with porous microspheres and nanoparticles, using an adsorption/infiltration process

    Directory of Open Access Journals (Sweden)

    Alcalá-Alcalá S

    2013-06-01

    Full Text Available Sergio Alcalá-Alcalá, Zaida Urbán-Morlán, Irene Aguilar-Rosas, David Quintanar-Guerrero Laboratorio de Investigación y Posgrado en Tecnología Farmacéutica, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, Cuautitlán Izcalli, Estado de México, México Abstract: A biodegradable polymeric system is proposed for formulating peptides and proteins. The systems were assembled through the adsorption of biodegradable polymeric nanoparticles onto porous, biodegradable microspheres by an adsorption/infiltration process with the use of an immersion method. The peptide drug is not involved in the manufacturing of the nanoparticles or in obtaining the microspheres; thus, contact with the organic solvent, interfaces, and shear forces required for the process are prevented during drug loading. Leuprolide acetate was used as the model peptide, and poly(D,L-lactide-co-glycolide (PLGA was used as the biodegradable polymer. Leuprolide was adsorbed onto different amounts of PLGA nanoparticles (25 mg/mL, 50 mg/mL, 75 mg/mL, and 100 mg/mL in a first stage; then, these were infiltrated into porous PLGA microspheres (100 mg by dipping the structures into a microsphere suspension. In this way, the leuprolide was adsorbed onto both surfaces (ie, nanoparticles and microspheres. Scanning electron microscopy studies revealed the formation of a nanoparticle film on the porous microsphere surface that becomes more continuous as the amount of infiltrated nanoparticles increases. The adsorption efficiency and release rate are dependent on the amount of adsorbed nanoparticles. As expected, a greater adsorption efficiency (~95% and a slower release rate were seen (~20% of released leuprolide in 12 hours when a larger amount of nanoparticles was adsorbed (100 mg/mL of nanoparticles. Leuprolide acetate begins to be released immediately when there are no infiltrated nanoparticles, and 90% of the peptide is released in the first 12 hours

  8. Functional characterization of TRAP1-like protein involved in modulating fibrotic processes mediated by TGF-β/Smad signaling in hypertrophic scar fibroblasts

    International Nuclear Information System (INIS)

    The transforming growth factor-β1 (TGF-β)-mediated signaling pathway is believed to be closely associated with wound healing and scar formation, in which TRAP1-like protein (TLP) plays a role in regulating the balance of Smad2 vs. Smad3 signaling. Our previous study revealed the relation between TLP and collagen synthesis in normal human skin fibroblasts. Here, we present a detailed analysis of the effects of TLP on the process of hypertrophic scar formation and contraction. To explore and verify a contribution of TLP to the pathological mechanism of hypertrophic scar fibroblasts (HSFb), we constructed lentiviral vectors that either overexpressed TLP or encoded small hairpin RNAs (shRNAs) targeting TLP, then we transfected them into HSFb. TLP knockdown in HSFb resulted in reduced levels of cell contraction, type I and type III collagen mRNA transcripts and protein expression, and higher levels of fibronectin (FN) compared to control groups. In addition, knockdown of TLP promoted the phosphorylation of Smad3 but repressed Smad2 and Erk-1/2 phosphorylation in human hypertrophic scar fibroblasts compared to control groups. The reduction of TLP did not interfere with HSF proliferative ability, but exogenous TLP cooperated with TGF-β1 to increase cell viability. Together, our findings demonstrate evidence for a contribution of TLP expression in hypertrophic scar formation and contraction. - Highlights: • TLP acted different roles in the activating of Smad2- and Smad3-dependent signaling. • TLP may induce TGF-β1-mediated collagens expression through Smad signalings and MAPK signaling. • TLP may enhance HSFb contraction by increasing the expression of α-SMA. • Exogenous TLP can cooperate with TGF-β1 to increase cell viability

  9. Functional characterization of TRAP1-like protein involved in modulating fibrotic processes mediated by TGF-β/Smad signaling in hypertrophic scar fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wang, X. [Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Department of Pediatric Surgery, Shanghai Children’s Medical Center, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200127 (China); Chu, J. [Department of Pediatric Surgery, Shanghai Children’s Medical Center, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200127 (China); Wen, C.J.; Fu, S.B.; Qian, Y.L. [Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Wo, Y. [Department of Anatomy, Institutes of Medical Sciences, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Wang, C., E-mail: wangchen2369@163.com [Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Wang, D.R., E-mail: wangdanru@126.com [Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China)

    2015-03-15

    The transforming growth factor-β1 (TGF-β)-mediated signaling pathway is believed to be closely associated with wound healing and scar formation, in which TRAP1-like protein (TLP) plays a role in regulating the balance of Smad2 vs. Smad3 signaling. Our previous study revealed the relation between TLP and collagen synthesis in normal human skin fibroblasts. Here, we present a detailed analysis of the effects of TLP on the process of hypertrophic scar formation and contraction. To explore and verify a contribution of TLP to the pathological mechanism of hypertrophic scar fibroblasts (HSFb), we constructed lentiviral vectors that either overexpressed TLP or encoded small hairpin RNAs (shRNAs) targeting TLP, then we transfected them into HSFb. TLP knockdown in HSFb resulted in reduced levels of cell contraction, type I and type III collagen mRNA transcripts and protein expression, and higher levels of fibronectin (FN) compared to control groups. In addition, knockdown of TLP promoted the phosphorylation of Smad3 but repressed Smad2 and Erk-1/2 phosphorylation in human hypertrophic scar fibroblasts compared to control groups. The reduction of TLP did not interfere with HSF proliferative ability, but exogenous TLP cooperated with TGF-β1 to increase cell viability. Together, our findings demonstrate evidence for a contribution of TLP expression in hypertrophic scar formation and contraction. - Highlights: • TLP acted different roles in the activating of Smad2- and Smad3-dependent signaling. • TLP may induce TGF-β1-mediated collagens expression through Smad signalings and MAPK signaling. • TLP may enhance HSFb contraction by increasing the expression of α-SMA. • Exogenous TLP can cooperate with TGF-β1 to increase cell viability.

  10. A novel signal transduction protein P(II) variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-P(II) complex formation.

    Science.gov (United States)

    Fokina, Oleksandra; Chellamuthu, Vasuki-Ranjani; Zeth, Kornelius; Forchhammer, Karl

    2010-06-11

    P(II) signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P(II) binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P(II) (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P(II) binding to NAGK, we searched for P(II) variants with altered binding characteristics and found P(II) variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P(II) protein. Analysis of interactions between these P(II) variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P(II) I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P(II) I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P(II) proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P(II) after having latched onto NAGK. Moreover, both P(II) I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of P(II)-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P(II) initiates the bending of the extended T-loop of P(II), followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex. PMID:20399792

  11. Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties

    OpenAIRE

    Maxime Saffon; Véronique Richard; Rafael Jiménez-Flores; Gauthier, Sylvie F.; Michel Britten; Yves Pouliot

    2013-01-01

    The objective of this study was to assess the impact of using heat-denatured whey:buttermilk protein aggregate in acid-set type yogurt production. Whey and buttermilk (25:75) protein concentrate was adjusted to pH 4.6, heated at 90 °C for 5 min, homogenized and freeze-dried. Set-type yogurts were prepared from skim milk standardized to 15% (w/v) total solids and 4.2% (w/v) protein using different levels of powdered skim milk or freeze-dried protein aggregate. The use of the protein aggregate ...

  12. An Algorithm for Finding Functional Modules and Protein Complexes in Protein-Protein Interaction Networks

    OpenAIRE

    Guangyu Cui; Yu Chen; De-Shuang Huang; Kyungsook Han

    2008-01-01

    Biological processes are often performed by a group of proteins rather than by individual proteins, and proteins in a same biological group form a densely connected subgraph in a protein-protein interaction network. Therefore, finding a densely connected subgraph provides useful information to predict the function or protein complex of uncharacterized proteins in the highly connected subgraph. We have developed an efficient algorithm and program for finding cliques and near-cliques in a prote...

  13. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... the vegetarian proteins, whether they have carbohydrate. Best Protein Choices The best choices are: Plant-based proteins ...

  14. A vector carrying the GFP gene (Green fluorescent protein as a yeast marker for fermentation processes Um vetor com o gene da GFP (Green fluorescent protein para a marcação de leveduras em processos fermentativos

    Directory of Open Access Journals (Sweden)

    Luiz Humberto Gomes

    2000-12-01

    Full Text Available Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3. Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.Leveduras contaminantes podem causar grandes perdas em processos fermentativos quando infectam culturas puras e degradam a qualidade do produto final. Estas leveduras podem ser detectadas por diversos métodos mas nenhum deles oferece resultados com a exatidão e precisão necessárias, quando os contaminantes estão em baixa freqüência. Culturas puras contendo um gene marcador podem ser utilizadas para a direta identificação de células e colônias contaminantes. Detecção rápida e fácil é desejada e marcadores morfológicos podem auxiliar na visualização da cultura marcada. O gene da GFP (green fluorescent protein extraído da Aequorea victoria

  15. The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

    OpenAIRE

    de Souza, Tiago Antonio; Soprano, Adriana Santos; de Lira, Nayara Patricia Vieira; Quaresma, Alexandre José Christino; Pauletti, Bianca Alves; Leme, Adriana Franco Paes; Benedetti, Celso Eduardo

    2012-01-01

    Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated...

  16. Behavior of Heat-Denatured Whey: Buttermilk Protein Aggregates during the Yogurt-Making Process and Their Influence on Set-Type Yogurt Properties

    Directory of Open Access Journals (Sweden)

    Maxime Saffon

    2013-09-01

    Full Text Available The objective of this study was to assess the impact of using heat-denatured whey:buttermilk protein aggregate in acid-set type yogurt production. Whey and buttermilk (25:75 protein concentrate was adjusted to pH 4.6, heated at 90 °C for 5 min, homogenized and freeze-dried. Set-type yogurts were prepared from skim milk standardized to 15% (w/v total solids and 4.2% (w/v protein using different levels of powdered skim milk or freeze-dried protein aggregate. The use of the protein aggregate significantly modified yogurt texture, but did not affect the water-holding capacity of the gel. Confocal laser-scanning microscope images showed the presence of large particles in milk enriched with protein aggregate, which directly affected the homogeneity of the clusters within the protein matrix. Thiol groups were freed during heating of the protein aggregate suspended in water, suggesting that the aggregates could interact with milk proteins during heating.

  17. From grain to feed – process development concerning production of high protein fractions from grain and legum products to be used in extruded fish feed pellets

    OpenAIRE

    Tolderlund Rasmussen, Hanne

    2009-01-01

    The Danish project “Organic Aquaculture” the link between sustainable production and superior products” is examining the availability of relevant organically produced crops with a high protein content to be used as raw materials for fish feed. Fish meal is the main source of protein for fish feed. On a global scale the production of fish meal will not increase. There is a demand for developing sustainable protein sources as a substitution for fish meal. Protein from crops and legumes is an ob...

  18. A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

    Directory of Open Access Journals (Sweden)

    Wang Zhikang

    2011-07-01

    at the possibility that these proteins may have been selected for their ability to process chloroplast pre-tRNAs with whole or partial CCA sequences. Our results also support the coevolution of tRNase Zs and tRNA 3'-trailer sequences in plants.

  19. The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I.

    Science.gov (United States)

    Colas des Francs-Small, Catherine; Falcon de Longevialle, Andéol; Li, Yunhai; Lowe, Elizabeth; Tanz, Sandra K; Smith, Caroline; Bevan, Michael W; Small, Ian

    2014-06-23

    Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two pentatricopeptide repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far. PMID:24958715

  20. The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I1[W][OPEN

    Science.gov (United States)

    Colas des Francs-Small, Catherine; Falcon de Longevialle, Andéol; Li, Yunhai; Lowe, Elizabeth; Tanz, Sandra K.; Smith, Caroline; Bevan, Michael W.; Small, Ian

    2014-01-01

    Pentatricopeptide repeat proteins constitute a large family of RNA-binding proteins in higher plants (around 450 genes in Arabidopsis [Arabidopsis thaliana]), mostly targeted to chloroplasts and mitochondria. Many of them are involved in organelle posttranscriptional processes, in a very specific manner. Splicing is necessary to remove the group II introns, which interrupt the coding sequences of several genes encoding components of the mitochondrial respiratory chain. The nad5 gene is fragmented in five exons, belonging to three distinct transcription units. Its maturation requires two cis- and two trans-splicing events. These steps need to be performed in a very precise order to generate a functional transcript. Here, we characterize two pentatricopeptide repeat proteins, ORGANELLE TRANSCRIPT PROCESSING439 and TANG2, and show that they are involved in the removal of nad5 introns 2 and 3, respectively. To our knowledge, they are the first two specific nad5 splicing factors found in plants so far. PMID:24958715

  1. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers. The...

  2. Analysis of Soybean Protein- Derived Peptides and the Effect of Cultivar, Environmental Conditions, and Processing on Lunasin Concentration in Soybean and Soy Products

    Science.gov (United States)

    Soybean, an important source of food proteins, has received increasing interest from the public because of its reported health benefits. These health benefits are attributed to its components including isoflavones, saponins, proteins and peptides. Lunasin, Bowman-Birk inhibitor, lectin, and ß-congly...

  3. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  4. Chronic treatment with a tryptophan-rich protein hydrolysate improves emotional processing, mental energy levels and reaction time in middle-aged women.

    Science.gov (United States)

    Mohajeri, M H; Wittwer, J; Vargas, K; Hogan, E; Holmes, A; Rogers, P J; Goralczyk, R; Gibson, E L

    2015-01-28

    Common pharmacological treatments of mood disorders aim to modulate serotonergic neurotransmission and enhance serotonin levels in the brain. Brain serotonin levels are dependent on the availability of its food-derived precursor essential amino acid tryptophan (Trp). We tested the hypothesis that delivery of Trp via food may serve as an alternative treatment, and examined the effects of a Trp-rich, bioavailable dietary supplement from egg protein hydrolysate on cognitive and emotional functions, mood state, and sleep quality. In a randomised, placebo-controlled, parallel trial, fifty-nine mentally and physically healthy women aged 45-65 years received placebo (n 30) or the supplement (n 29) (both as 0.5 g twice per d) for 19 d. Emotional processing was significantly changed by supplementation, exhibiting a shift in bias away from negative stimuli. The results for the Affective Go/No-Go Task exhibited a slowing of responses to negative words, suggesting reduced attention to negative emotional stimuli. The results for the Facial Emotional Expression Rating Task also supported a shift away from attention to negative emotions and a bias towards happiness. An increase in arousal-like symptoms, labelled 'high energy', shorter reaction times and a slight benefit to sustained attention were observed in the treated subjects. Finally, when the supplement was taken 60-90 min before bedtime, a feeling of happiness before going to bed was consistently reported. In summary, daily consumption of a low-dose supplement containing bioavailable Trp may have beneficial effects on emotional and cognitive functions. PMID:25572038

  5. The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter

    OpenAIRE

    Nagai, Hiroki; Roy, Craig R.

    2001-01-01

    Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted Do...

  6. Regulating of various physiological and pathological processes by human GIT1/2 proteins%人GIT1/2蛋白调控多种生理和病理进程

    Institute of Scientific and Technical Information of China (English)

    韦超; 贺福初; 王建

    2012-01-01

    G蛋白偶联受体激酶相互作用蛋白1/2 [G-protein-coupled receptor (GPCR) kinase interacting protein 1/2,GIT1/2]属于多结构域蛋白质.它们参与多种细胞生物学过程:(1)通过其ARF GTPase-激活蛋白(ARF GTPase-activating protein,ARF-GAP)活性调节细胞骨架动态变化;(2)参与G蛋白偶联受体的内化及细胞膜转运;(3)作为支架蛋白调控信号转导.GIT蛋白家族在骨代谢、血管稳态、神经发育以及肿瘤等生理或病理条件下发挥关键的调控作用.本文着重就近年来GIT蛋白家族在生理与病理方面的调控作用作一综述.%The G-protein-coupled receptor (GPCR) kinase interacting protein 1 and 2 (GIT1 and GIT2) are multidomain proteins involved in diverse cellular processes, such as regulation of cytoskeleton dynamics by their ARF-GAP activity, modulation of receptor internalization and membrane trafficking, as well as controlling signaling transduction. The diversity of their functions plays significant roles during physiological and pathological conditions, such as bone metabolism, vascular stability, neuron development and cancer. Here we summarize the current understanding of GIT protein family in physiological and pathological processes.

  7. Challenges of protein extraction from recalcitrant plant tissues for proteomics

    Science.gov (United States)

    Proteins play an important role in several biological processes. Proteomics encompasses basically four principal applications, namely protein mining, protein expression profiling, protein-network mapping and mapping of protein modifications. The results in these applications depend mostly on the c...

  8. Dietary supplementation with dried chicory root triggers changes in the blood serum proteins engaged in the clotting process and the innate immune response in growing pigs.

    Science.gov (United States)

    Lepczynski, A; Herosimczyk, A; Ozgo, M; Skomial, J; Taciak, M; Barszcz, M; Berezecka, N

    2015-02-01

    The aim of the study was to characterize the systemic immune and metabolic alterations in the blood serum of growing pigs in response to a dietary supplementation with 4% of dried chicory roots. This was achieved by examining the influence of the experimental diet on serum protein changes especially these related with immunology and lipid metabolism. Serum proteins with the isoelectric point ranging from pH 3.0 to 10.0 were separated using high resolution two-dimensional electrophoresis. As a result, we found that experimental diet triggered significant changes in 37 protein spots. Of these, 14 were up-regulated, whereas 23 showed down-regulation. Of 37 significantly altered protein spots, 24 were successfully identified, representing 14 distinct gene products. Implementation of the dried chicory roots into the diet of growing pigs caused a significant down-regulation of apolipoprotein C-II complement component C6, C-reactive protein, CD14 antigen, C4b binding protein α and β chains, and fibrinogen. Piglets fed experimental diet had similar IgA, IgG and IgM concentrations, although the level of IgM tended to be lower compared to the control group. It is concluded that diet supplemented with 4% of dried chicory root may exert anti-inflammatory properties and affect lipid metabolism in growing pigs. PMID:25716964

  9. A PPR protein in the PLS subfamily stabilizes the 5'-end of processed rpl16 mRNAs in maize chloroplasts.

    Science.gov (United States)

    Hammani, Kamel; Takenaka, Mizuki; Miranda, Rafael; Barkan, Alice

    2016-05-19

    Pentatricopeptide repeat (PPR) proteins are a large family of helical-repeat proteins that bind RNA in mitochondria and chloroplasts. Precise RNA targets and functions have been assigned to only a small fraction of the >400 members of the PPR family in plants. We used the amino acid code governing the specificity of RNA binding by PPR repeats to infer candidate-binding sites for the maize protein PPR103 and its ortholog Arabidopsis EMB175. Genetic and biochemical data confirmed a predicted binding site in the chloroplast rpl16 5'UTR to be a site of PPR103 action. This site maps to the 5' end of transcripts that fail to accumulate in ppr103 mutants. A small RNA corresponding to the predicted PPR103 binding site accumulates in a PPR103-dependent fashion, as expected of PPR103's in vivo footprint. Recombinant PPR103 bound specifically to this sequence in vitro These observations imply that PPR103 stabilizes rpl16 mRNA by impeding 5'→3' RNA degradation. Previously described PPR proteins with this type of function consist of canonical PPR motifs. By contrast, PPR103 is a PLS-type protein, an architecture typically associated with proteins that specify sites of RNA editing. However, PPR103 is not required to specify editing sites in chloroplasts. PMID:27095196

  10. A PPR protein in the PLS subfamily stabilizes the 5′-end of processed rpl16 mRNAs in maize chloroplasts

    Science.gov (United States)

    Hammani, Kamel; Takenaka, Mizuki; Miranda, Rafael; Barkan, Alice

    2016-01-01

    Pentatricopeptide repeat (PPR) proteins are a large family of helical-repeat proteins that bind RNA in mitochondria and chloroplasts. Precise RNA targets and functions have been assigned to only a small fraction of the >400 members of the PPR family in plants. We used the amino acid code governing the specificity of RNA binding by PPR repeats to infer candidate-binding sites for the maize protein PPR103 and its ortholog Arabidopsis EMB175. Genetic and biochemical data confirmed a predicted binding site in the chloroplast rpl16 5′UTR to be a site of PPR103 action. This site maps to the 5′ end of transcripts that fail to accumulate in ppr103 mutants. A small RNA corresponding to the predicted PPR103 binding site accumulates in a PPR103-dependent fashion, as expected of PPR103's in vivo footprint. Recombinant PPR103 bound specifically to this sequence in vitro. These observations imply that PPR103 stabilizes rpl16 mRNA by impeding 5′→3′ RNA degradation. Previously described PPR proteins with this type of function consist of canonical PPR motifs. By contrast, PPR103 is a PLS-type protein, an architecture typically associated with proteins that specify sites of RNA editing. However, PPR103 is not required to specify editing sites in chloroplasts. PMID:27095196

  11. Absolute nutrient concentration measurements in cell culture media: (1)H q-NMR spectra and data to compare the efficiency of pH-controlled protein precipitation versus CPMG or post-processing filtering approaches.

    Science.gov (United States)

    Goldoni, Luca; Beringhelli, Tiziana; Rocchia, Walter; Realini, Natalia; Piomelli, Daniele

    2016-09-01

    The NMR spectra and data reported in this article refer to the research article titled "A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using q-NMR" [1]. We provide the (1)H q-NMR spectra of cell culture media (DMEM) after removal of serum proteins, which show the different efficiency of various precipitating solvents, the solvent/DMEM ratios, and pH of the solution. We compare the data of the absolute nutrient concentrations, measured by PULCON external standard method, before and after precipitation of serum proteins and those obtained using CPMG (Carr-Purcell-Meiboom-Gill) sequence or applying post-processing filtering algorithms to remove, from the (1)H q-NMR spectra, the proteins signal contribution. For each of these approaches, the percent error in the absolute value of every measurement for all the nutrients is also plotted as accuracy assessment. PMID:27331118

  12. Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitates expression of diverse tissue-specific isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Parra, Marilyn; Gee, Sherry; Chan, Nadine; Ryaboy, Dmitriy; Dubchak, Inna; Narla, Mohandas; Gascard, Philippe D.; Conboy, John G.

    2004-07-15

    The EPB41 (protein 4.1) genes epitomize the resourcefulness of the mammalian genome to encode a complex proteome from a small number of genes. By utilizing alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing, EPB41, EPB41L2, EPB41L3, and EPB41L1 encode a diverse array of structural adapter proteins. Comparative genomic and transcript analysis of these 140kb-240kb genes indicates several unusual features: differential evolution of highly conserved exons encoding known functional domains, interspersed with unique exons whose size and sequence variations contribute substantially to intergenic diversity: alternative first exons, most of which map far upstream of the coding regions; and complex tissue-specific alternative pre-mRNA splicing that facilitates synthesis of functionally different complements of 4.1 proteins in various cells. Understanding the splicing regulatory networks that control protein 4.1 expression will be critical to a full appreciation of the many roles of 4.1 proteins in normal cell biology and their proposed roles in human cancer.

  13. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    Science.gov (United States)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  14. Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

    International Nuclear Information System (INIS)

    The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6122, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 Å, and contained two molecules in the asymetric unit. It diffracted to 2.24 Å resolution

  15. Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta). II. An in vitro study of the transport process and the influence of temperature.

    Science.gov (United States)

    Slot, J W; Geuze, J J

    1976-03-16

    Frog pancreatic tissue was pulse-labelled in vitro with 3H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions. Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae. Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues. The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30 degrees C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog. PMID:1083293

  16. : Protein flexibility

    OpenAIRE

    Bornot, Aurélie; Offmann, Bernard; De Brevern, Alexandre

    2007-01-01

    Protein structures and protein structural models are great tools to reach protein function and provide very relevant information for drug design. Nevertheless, protein structures are not rigid entities. Cutting-edge bioinformatics methods tend to take into account the flexibility of these macromolecules. We present new approaches used to define protein structure flexibility.

  17. Immunological analysis of potato leafroll luteovirus (PLRV) P1 expression identifies a 25 kDa RNA-binding protein derived via P1 processing.

    OpenAIRE

    Prüfer, D; Kawchuk, L; Monecke2, M; Nowok, S; Fischer, R.; Rohde, W

    1999-01-01

    Mono- and polyclonal antibodies directed against different domains of the potato leafroll luteovirus (PLRV) P1 (ORF1) protein were applied to the analysis of P1 expression during PLRV replication in planta. Western analyses detected P1 and a protein of approximately 25 kDa (P1-C25) that accumulated to readily detectable amounts in PLRV-infected plants, but was not detected by in vitro cell-free translation of P1. P1-C25 represents the C-terminus of P1 and is a proteolytic cleavage product pro...

  18. Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing.

    OpenAIRE

    Hackstadt, T; Messer, R; Cieplak, W; Peacock, M G

    1992-01-01

    The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus o...

  19. Direct Repeat 6 from human herpesvirus-6B encodes a nuclear protein that forms a complex with the viral DNA processivity factor p41

    OpenAIRE

    Schleimann, Mariane H; Møller, Janni M. L.; Emil Kofod-Olsen; Per Höllsberg

    2009-01-01

    The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited ...

  20. Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 participate in the process of catabolite inactivation of maltose permease

    Czech Academy of Sciences Publication Activity Database

    Mayordomo, I.; Regelmann, J.; Horák, Jaroslav; Sanz, P.

    2003-01-01

    Roč. 544, 1-3 (2003), s. 160-164. ISSN 0014-5793 Grant ostatní: Spanish Ministry of Educaton and Science(ES) PB98-0486 Institutional research plan: CEZ:AV0Z5011922 Keywords : 14-3-3 proteins * maltose permease * catabolite inactivation Subject RIV: CE - Biochemistry Impact factor: 3.609, year: 2003

  1. Total protein

    Science.gov (United States)

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  2. Antigen Processing of the Heptavalent Pneumococcal Conjugate Vaccine Carrier Protein CRM197 Differs Depending on the Serotype of the Attached Polysaccharide

    OpenAIRE

    Leonard, Ethan G.; Canaday, David H.; Harding, Clifford V.; Schreiber, John R.

    2003-01-01

    The pneumococcal (Pn) conjugate vaccine includes seven different polysaccharides (PS) conjugated to CRM197. Utilizing antigen-processing cells and a CRM197-specific mouse T-cell hybridoma, we found that the serotype of conjugated PnPS dramatically affected antigen processing of CRM197. Unconjugated CRM197 and serotype conjugates 14 and 18C were processed more efficiently.

  3. G Protein-coupled receptors

    OpenAIRE

    Ross, Elliott M.

    2014-01-01

    G protein-coupled receptors and heterotrimeric G proteins can diffuse laterally in the plasma membrane such that one receptor can catalyze the activation (GDP/GTP exchange) of multiple G proteins. In some cases, these processes are fast enough to support molecular signal amplification, where a single receptor maintains the activation of multiple G proteins at steady-state. Amplification in cells is probably highly regulated. It depends upon the identities of the G receptor and G protein - som...

  4. Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing.

    Science.gov (United States)

    Hackstadt, T; Messer, R; Cieplak, W; Peacock, M G

    1992-01-01

    The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120- and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickettsii. The 168-kDa putative precursor of the avirulent strain of R. rickettsii was not extracted from the surface by dilute buffers, as is the 120-kDa protein of virulent R. rickettsii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain. Images PMID:1729180

  5. Protein (Cyanobacteria): 28423 [

    Lifescience Database Archive (English)

    Full Text Available ZP_10226597.1 1117:517 1118:7626 1125:2051 1160279:627 Type 4 prepilin-like proteins leader ... pept ... ide-processing enzyme (Includes: Leader ... peptidase ; N-methyltransferase) Microcystis sp. T ...

  6. Protein Colloidal Aggregation Project

    Science.gov (United States)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  7. Process of peanut protein milk powder by dry preparation%花生蛋白奶粉的干法生产工艺研究

    Institute of Scientific and Technical Information of China (English)

    章宝; 单杨; 李高阳

    2011-01-01

    以去壳花生仁为原料,经低温烘烤、脱红衣、冷榨脱脂、超微粉碎等工艺生产出花生蛋白粉,将其与全脂奶粉混合生产花生蛋白奶粉,所得产品溶解度高、冲调性好、口感细腻、营养均衡,同时具有奶味和花生特有的香味。%Peanut protein milk powder was made by mixing whole milk powder with peanut protein powder,which was made by the technologies of low-temperature baking,taking the peanut red skin off,cold-pressed degreasing and ultra-fine pulverizatio by using shelled pea

  8. Interfacial Protein-Protein Associations

    OpenAIRE

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2013-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for ...

  9. Korean red ginseng extract rejuvenates testicular ineffectiveness and sperm maturation process in aged rats by regulating redox proteins and oxidative defense mechanisms.

    Science.gov (United States)

    Kopalli, Spandana Rajendra; Hwang, Seock-Yeon; Won, Yu-Jin; Kim, Sung-Won; Cha, Kyu-Min; Han, Chang-Kyun; Hong, Jae-Yup; Kim, Si-Kwan

    2015-09-01

    Distortion of intracellular oxidant and antioxidant balances appears to be a common feature that underlies in age-related male sexual impairment. Therefore regulating oxidative defense mechanisms might be an ideal approach in improving male sexual dysfunctions. In the present study, the effect of Korean red ginseng aqueous extract (KRG) on age-induced testicular dysfunction in rats was investigated. KRG (200mg/kg) mixed with regular pellet diet was administered orally for six months and the morphological, spermatogenic and antioxidant enzyme status in testis of aged rats (18months) were evaluated. Data indicated a significant change in morphology and decrease in spermatogenesis-related parameters in aged rats (AC) compared with young rats (YC). Sperm number, germ cell count, Sertoli cell count and Sertoli cell index were significantly (p<0.05) restored in KRG-treated aged rat groups (G-AC). Further the increased lipid peroxidation as measured by malondialdehyde (p<0.05), and altered enzymatic (superoxide dismutase, glutathione peroxidase, glutathione S-transferase, glutathione reductase and catalase) and non-enzymatic (reduced glutathione, ascorbic acid and α-tocopherol) antioxidants (p<0.05) were attenuated by KRG treatment in aged rats to near normal levels as in YC groups. Furthermore, proteomic analysis demonstrated differential expression of selected proteins such as phosphatidylinositol transfer protein, fatty acid binding protein-9, triosephosphate isomerase-1 and aldehyde (aldose) reductase-1in aged rats was significantly (p<0.05) protected by KRG treatment. In conclusion, long-term administration of KRG restored aging-induced testicular ineffectiveness in rats by modulating redox proteins and oxidative defense mechanisms. PMID:25980653

  10. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-05-01

    Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. PMID:26472128

  11. Protein - Which is Best?

    Science.gov (United States)

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  12. Study of factors that interfere in the labelling process of erythrocytes and plasma proteins with Technetium-99m; Estudo de fatores que interferem no processo de marcacao de hemacias e proteinas plasmaticas com tecnecio-99m

    Energy Technology Data Exchange (ETDEWEB)

    Gutfilen, Bianca

    1989-12-31

    The labelling of red blood cells (RBC) with technetium-99m (Tc-99m) depends on several factors, as the stannous ion (Sn++) concentration, time, temperature, the presence of plasma proteins (PP) and others. However the Sn++ concentration seems to be the most important factor; probably because the uptake of this reducing agent by RBC is limited. The excess of Sn++ in extracellular medium can determine the labelling of PP. the modifications of RBC at 50 deg C described in the literature, the possibility of labelling RBC with Tc-99m at this temperature and experimental results obtained made it possible to perform spleen selective scintigraphy through a simple technique with few manipulations. The effect of gentamicin, nifedipine and verapamil in the labelling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca++ and Sn++. The results show that, under some conditions, these drugs are capable to alter this Tc-99m incorporation. The modification of the ionic distribution determined by these drugs or the blockage of Sn++ and/or Tc-99m or the fact that they bind theirselves to plasma proteins, or the possibility of the labelling of these drugs, are factors that can interfere in the labelling process of red blood cells and plasma proteins with Tc-99m. (author) 55 refs., 12 figs., 5 tabs.

  13. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    OpenAIRE

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

  14. c-IAP1 Binds and Processes PCSK9 Protein: Linking the c-IAP1 in a TNF-α Pathway to PCSK9-Mediated LDLR Degradation Pathway

    Directory of Open Access Journals (Sweden)

    David Hornby

    2012-10-01

    Full Text Available Recent genetic studies have shown that PCSK9, one of the key genes in cholesterol metabolism, plays a critical role by controlling the level of low-density lipoprotein receptor. However, how PCSK9 mediates LDLR degradation is still unknown. By combining a shotgun proteomic method and differential analysis of natural occurring mutations of the PCSK9 gene, we found that an E3 ubiquitin ligase c-IAP1 binds and processes PCSK9 protein. One of the ‘gain-of-function’ mutations, S127R, is defective with respect to binding to c-IAP1, and thus has defective autocatalytic activity. Knockdown of c-IAP1 impairs PCSK9 processing and autocatalytic cleavage. In c-IAP1 null mouse embryonic fibroblasts (MEFs, there is a dramatic decrease in secreted mature PCSK9 protein accompanied by a significant increase in LDLR protein levels compared with matched wild-type MEF cells. c-IAP1 also acts as an E3 ligase for ubiquitination of PCSK9. Ubiquitin containing only lysine-27 mediated PCSK9 ubiquitination by c-IAP1. Given K27-linked polyubiquitination promotes lysosomal localization, the finding indicates the c-IAP1 acts on both secretion of PCSK9 and its lysosomal localization. The novel pathway described here will open new avenues for exploring novel disease treatments.

  15. Comparison of Soy Protein Dope with Yeast Protein Dope on the Rheological Properties

    OpenAIRE

    Hayakawa, Isao; Chang, Hung Min; Katoh, Tatsuo

    1984-01-01

    Rheological properties of isolated soybean protein dopes were compared with those of yeart protein dopes in order to find out their application and processing. The elastic properties of soybean protein dope were better than those of yeast protein dope prepared with high protein concentrates because the viscoelastic absorption of soybean protein dope was smaller than that of yeast protein dope and the capacity of water holding was higher than that of yeast protein dope. On the other hand, yeas...

  16. Regulated protein aggregation: stress granules and neurodegeneration

    OpenAIRE

    Wolozin Benjamin

    2012-01-01

    Abstract The protein aggregation that occurs in neurodegenerative diseases is classically thought to occur as an undesirable, nonfunctional byproduct of protein misfolding. This model contrasts with the biology of RNA binding proteins, many of which are linked to neurodegenerative diseases. RNA binding proteins use protein aggregation as part of a normal regulated, physiological mechanism controlling protein synthesis. The process of regulated protein aggregation is most evident in formation ...

  17. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Amarnath Chtterjee; Ashutosh Kumar; Jeetender Chugh; Sudha Srivastava; Neel S Bhavesh; Ramakrishna V Hosur

    2005-01-01

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end.

  18. Protein Models Comparator

    CERN Document Server

    Widera, Paweł

    2011-01-01

    The process of comparison of computer generated protein structural models is an important element of protein structure prediction. It has many uses including model quality evaluation, selection of the final models from a large set of candidates or optimisation of parameters of energy functions used in template free modelling and refinement. Although many protein comparison methods are available online on numerous web servers, their ability to handle a large scale model comparison is often very limited. Most of the servers offer only a single pairwise structural comparison, and they usually do not provide a model-specific comparison with a fixed alignment between the models. To bridge the gap between the protein and model structure comparison we have developed the Protein Models Comparator (pm-cmp). To be able to deliver the scalability on demand and handle large comparison experiments the pm-cmp was implemented "in the cloud". Protein Models Comparator is a scalable web application for a fast distributed comp...

  19. The RNA Binding Zinc Finger Protein Tristetraprolin Regulates AU-Rich mRNAs Involved in Breast Cancer-Related Processes

    OpenAIRE

    Al-Souhibani, Norah; Al-Ahmadi, Wijdan; Hesketh, John E.; Blackshear, Perry J.; Khabar, Khalid S.A.

    2010-01-01

    Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc finger RNA binding protein that regulates the stability of certain AU-rich mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared to normal cell types. Here we found that TTP expression was lower in invasive breast cancer cells (MDA-MB-231) compared to normal breast cell lines, MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc finger TTP mutant that act as dominant negati...

  20. 重组碎羊肉加工中非肉蛋白的选择%Selection of Non-Meat Proteins for the Process of Restructured Mutton

    Institute of Scientific and Technical Information of China (English)

    梁海燕; 王国昌

    2012-01-01

    在碎羊肉中添加质量分数为0.04%的转谷氨酰胺酶(TG),再分别添加质量分数均为0、0.2%、0.4%、0.6%、0.8%、1.0%的卵清蛋白、蛋黄蛋白、乳清蛋白组成3个试验组.将其在6℃下作用138 min,经过成形、解冻后测定其保形性,并用扫描电子显微镜(SEM)观察其微观结构.对SEM扫描结果分析可知,TG的加入可使碎羊肉形成致密的凝胶网络结构,适量地添加非肉蛋白有利于提高碎肉制品的保形性,其中质量分数为0.4%的蛋黄蛋白提高碎羊肉保形性的效果较好.%After 0.04% (mass ratio) transglutaminase(TG) was added into minced mutton, ovalbumin, yolk protein and whey protein was added at the mass ratio of 0, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% respectively, and reacting at 6℃ for 138 min. The samples were frozen and thawed to test the binding properties. And the microslruclure was observed by scanning election microscope (SEM). The result showed that 0.4% yolk protein could increase the binding property of the minced mutton the best. According to SEM, the addition of TG could help the formation of uniform gel network in minced mutton; and the binding property was improved by the addition of non-meat proteins.