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Sample records for autoinducer-2 processing protein

  1. The crystal structure of the Escherichia coli autoinducer-2 processing protein LsrF.

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    Zamia Diaz

    2009-08-01

    Full Text Available Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2. Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (alphabeta(8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology with aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase.

  2. 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.

  3. Autoinducer-2 Quorum Sensing Contributes to Regulation of Microcin PDI in Escherichia coli

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    Shao-Yeh Lu

    2017-12-01

    Full Text Available The Escherichia coli quorum sensing (QS signal molecule, autoinducer-2 (AI-2, reaches its maximum concentration during mid-to-late growth phase after which it quickly degrades during stationary phase. This pattern of AI-2 concentration coincides with the up- then down-regulation of a recently described microcin PDI (mccPDI effector protein (McpM. To determine if there is a functional relationship between these systems, a prototypical mccPDI-expressing strain of E. coli 25 was used to generate ΔluxS, ΔlsrACDBFG (Δlsr, and ΔlsrR mutant strains that are deficient in AI-2 production, transportation, and AI-2 transport regulation, respectively. Trans-complementation, RT-qPCR, and western blot assays were used to detect changes of microcin expression and synthesis under co-culture and monoculture conditions. Compared to the wild-type strain, the AI-2-deficient strain (ΔluxS and -uptake negative strain (Δlsr were >1,000-fold less inhibitory to susceptible bacteria (P < 0.05. With in trans complementation of luxS, the AI-2 deficient mutant reduced the susceptible E. coli population by 4-log, which was within 1-log of the wild-type phenotype. RT-qPCR and western blot results for the AI-2 deficient E. coli 25 showed a 5-fold reduction in mcpM transcription with an average 2-h delay in McpM synthesis. Furthermore, overexpression of sRNA micC and micF (both involved in porin protein regulation was correlated with mcpM regulation, consistent with a possible link between QS and mcpM regulation. This is the direct first evidence that microcin regulation can be linked to quorum sensing in a Gram-negative bacterium.

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

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    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.

  5. 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

    Bacterial strains of the species Arthrobacter nicotianae, Corynebacterium ammoniagenes, Corynebacterium casei, Microbacterium barkeri, Microbacterium gubbeenense and Staphylococcus equorum subsp. linens, all isolated from the smear of surface ripened cheeses, were found to possess autoinducer-2 (...... increased by dairy-relevant stress conditions, indicates that AI-2 signalling might be important in regulation of microbial succession during ripening of surface ripened cheeses.......Bacterial strains of the species Arthrobacter nicotianae, Corynebacterium ammoniagenes, Corynebacterium casei, Microbacterium barkeri, Microbacterium gubbeenense and Staphylococcus equorum subsp. linens, all isolated from the smear of surface ripened cheeses, were found to possess autoinducer-2 (AI......-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...

  6. Chemorepulsion from the Quorum Signal Autoinducer-2 Promotes Helicobacter pylori Biofilm Dispersal

    OpenAIRE

    Anderson, Jeneva K.; Huang, Julie Y.; Wreden, Christopher; Sweeney, Emily Goers; Goers, John; Remington, S. James; Guillemin, Karen

    2015-01-01

    ABSTRACT The gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H.?pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H.?pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (?luxS strains) or are defective for chemotaxis (?cheA strains) formed more spatially homogenous biofilms with a...

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

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

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    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.

  9. Autoinducer 2 Signaling via the Phosphotransferase FruA Drives Galactose Utilization by Streptococcus pneumoniae, Resulting in Hypervirulence

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    Claudia Trappetti

    2017-01-01

    Full Text Available Communication between bacterial cells is crucial for the coordination of diverse cellular processes that facilitate environmental adaptation and, in the case of pathogenic species, virulence. This is achieved by the secretion and detection of small signaling molecules called autoinducers, a process termed quorum sensing. To date, the only signaling molecule recognized by both Gram-positive and Gram-negative bacteria is autoinducer 2 (AI-2, synthesized by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase as a by-product of the activated methyl cycle. Homologues of LuxS are ubiquitous in bacteria, suggesting a key role in interspecies, as well as intraspecies, communication. Gram-negative bacteria sense and respond to AI-2 via the Lsr ABC transporter system or by the LuxP/LuxQ phosphorelay system. However, homologues of these systems are absent from Gram-positive bacteria and the AI-2 receptor is unknown. Here we show that in the major human pathogen Streptococcus pneumoniae, sensing of exogenous AI-2 is dependent on FruA, a fructose-specific phosphoenolpyruvate-phosphotransferase system that is highly conserved in Gram-positive pathogens. Importantly, AI-2 signaling via FruA enables the bacterium to utilize galactose as a carbon source and upregulates the Leloir pathway, thereby leading to increased production of capsular polysaccharide and a hypervirulent phenotype.

  10. Production, detection and application perspectives of quorum sensing autoinducer-2 in bacteria.

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    Zhao, Jing; Quan, Chunshan; Jin, Liming; Chen, Ming

    2018-02-20

    Autoinducer-2 (AI-2) is a major signal molecule in bacterial quorum sensing (QS) besides N-acyl homoserine lactones (AHLs or AI-1). AI-2 mediated QS pathways have been proved to regulate gene expression and physiological behaviors of bacteria in either intraspecies or interspecies communication. Recent reviews have mainly summarized AI-2 structures, AI-2 mediated QS pathways and the role of AI-2 in gene regulation, etc. In this article, we present a comprehensive review of AI-2 production, detection and applications. Firstly, intracellular AI-2 synthetic routes were outlined and environmental influences on AI-2 production were focused. Furthermore, recent advances in AI-2 detection and quantification were elucidated from an overall perspective. An in-depth understanding of mechanisms and features of various detection methods may facilitate development of new technologies aimed at signal molecule detection. Finally, utilization of AI-2 mediated QS in health improvement, water treatment and drug production indicate promising and extensive application perspectives of QS strategies. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Autoinducer-2 Facilitates Pseudomonas aeruginosa PAO1 Pathogenicity in Vitro and in Vivo

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    Hongdong Li

    2017-10-01

    Full Text Available Bacterial communication systems, such as quorum sensing (QS, have provided new insights of alternative approaches in antimicrobial treatment. We recently reported that one QS signal, named as autoinducer-2 (AI-2, can affect the behaviors of Pseudomonas aeruginosa PAO1 in a dose-dependent manner. In this study, we aimed to investigate the effects of AI-2 on P. aeruginosa PAO1 biofilm formation and virulence factors production in vitro, and in vivo using a pulmonary infection mouse model. Exogenous AI-2 resulted in increased biofilms architecture, the number of viable cells, and the yield of pyocyanin and elastase virulence factors in wild type P. aeruginosa PAO1. However, no such effect was observed in P. aeruginosa lasR rhlR mutant strain. In vivo, the use of AI-2 significantly increased the mortality, lung bacterial count and histological lung damage of mice with acute P. aeruginosa PAO1 infection. Our data suggest that AI-2 promotes the formation of P. aeruginosa PAO1 biofilms and the production of virulence factors by interfering with P. aeruginosa QS systems, resulting in decreased host survival. AI-2 may be a therapeutic target for the clinical treatment of a co-infection of P. aeruginosa and AI-2 producing bacteria.

  12. New bicyclic brominated furanones as potent autoinducer-2 quorum-sensing inhibitors against bacterial biofilm formation.

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    Park, Ji Su; Ryu, Eun-Ju; Li, Linzi; Choi, Bong-Kyu; Kim, B Moon

    2017-09-08

    Bacterial behaviors such as virulence factor secretion and biofilm formation are critical for survival, and are effectively regulated through quorum sensing, a mechanism of intra- and interspecies communication in response to changes in cell density and species complexity. Many bacterial species colonize host tissues and form a defensive structure called a biofilm, which can be the basis of inflammatory diseases. Periodontitis, a chronic inflammatory disease affecting the periodontium, is caused by subgingival biofilms related to periodontopathogens. In particular, Fusobacterium nucleatum is a major co-aggregation bridge organism in the formation and growth of subgingival biofilms, linking the early and late colonizers in periodontal biofilms. According to our previous study, the intergeneric quorum-sensing signal molecule autoinducer-2 (AI-2) of F. nucleatum plays a key role in intra- and interspecies interactions of periodontopathogens, and may be a good target for periodontal biofilm inhibition. Recently, brominated furanones produced by the macroalga Delisea pulchra were shown to inhibit biofilm formation via AI-2, and have been investigated toward the goal of increasing the inhibition effect. In this study, we describe the synthesis of new bromofuranone analogs, i.e., 3-(dibromomethylene)isobenzofuran-1(3H)-one derivatives, and demonstrate their inhibitory activities against biofilm formation by periodontopathogens, including F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Evaluation of two autoinducer-2 quantification methods for application in marine environments

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    Wang, Tian-Nyu

    2018-02-11

    This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also investigated the stability of AI-2 in varying pH, temperature and media, as well as quantified the amount of AI-2 signals in marine samples.HPLC-FLD method showed a higher level of reproducibility and precision compared to V. harveyi BB170 bioassay. Alkaline pH > 8 and high temperature (> 37°C) increased the instability of AI-2. The AI-2 concentrations in seawater were low, ca. 3.2-27.6 pmol l-1 whereas 8- week old marine biofilm grew on an 18.8 cm2 substratum accumulated ca. 0.207 nmol of AI-2.Both methods have pros and cons for AI-2 quantification in marine samples. Regardless, both methods reported a ubiquitous presence of AI-2 in both planktonic and biomass fractions of seawater, as well as in marine biofilm.In this study, AI-2 signals were for the first time enumerated in marine samples to reveal the ubiquitous presence of AI-2 in this environment. The findings suggest a possible role of AI-2 in biofilm formation in marine environment, and the contribution of AI-2 in biofilm-associated problems such as biofouling and biocorrosion. This article is protected by copyright. All rights reserved.

  14. Differential effect of autoinducer 2 of Fusobacterium nucleatum on oral streptococci.

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    Jang, Yun-Ji; Sim, Jaehyun; Jun, Hye-Kyoung; Choi, Bong-Kyu

    2013-11-01

    Autoinducer 2 (AI-2) is a quorum sensing molecule and plays an important role in dental biofilm formation, mediating interspecies communication and virulence expression of oral bacteria. Fusobacterium nucleatum connects early colonizing commensals and late colonizing periodontopathogens. F. nucleatum AI-2 and quorum sensing inhibitors (QSIs) can manipulate dental biofilm formation. In this study, we evaluated the effect of F. nucleatum AI-2 and QSIs on biofilm formation of Streptococcus gordonii and Streptococcus oralis, which are initial colonizers in dental biofilm. F. nucleatum AI-2 significantly enhanced biofilm growth of S. gordonii and attachment of F. nucleatum to preformed S. gordonii biofilms. By contrast, F. nucleatum AI-2 reduced biofilm growth of S. oralis and attachment of F. nucleatum to preformed S. oralis biofilms. The QSIs, (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone and d-ribose, reversed the stimulatory and inhibitory effects of AI-2 on S. gordonii and S. oralis, respectively. In addition, co-culture using a two-compartment system showed that secreted molecules of F. nucleatum had the same effect on biofilm growth of the streptococci as AI-2. Our results demonstrate that early colonizing bacteria can influence the accretion of F. nucleatum, a secondary colonizer, which ultimately influences the binding of periodontopathogens. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

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

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    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. Copyright © 2016. Published by Elsevier B.V.

  16. Staphylococcus aureus autoinducer-2 quorum sensing decreases biofilm formation in an icaR-dependent manner

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    Yu Dan

    2012-12-01

    Full Text Available Abstract Background Staphylococcus aureus is an important pathogen that causes biofilm-associated infection in humans. Autoinducer 2 (AI-2, a quorum-sensing (QS signal for interspecies communication, has a wide range of regulatory functions in both Gram-positive and Gram-negative bacteria, but its exact role in biofilm formation in S. aureus remains unclear. Results Here we demonstrate that mutation of the AI-2 synthase gene luxS in S. aureus RN6390B results in increased biofilm formation compared with the wild-type (WT strain under static, flowing and anaerobic conditions and in a mouse model. Addition of the chemically synthesized AI-2 precursor in the luxS mutation strain (ΔluxS restored the WT phenotype. Real-time RT-PCR analysis showed that AI-2 activated the transcription of icaR, a repressor of the ica operon, and subsequently a decreased level of icaA transcription, which was presumably the main reason why luxS mutation influences biofilm formation. Furthermore, we compared the roles of the agr-mediated QS system and the LuxS/AI-2 QS system in the regulation of biofilm formation using the ΔluxS strain, RN6911 and the Δagr ΔluxS strain. Our data indicate a cumulative effect of the two QS systems on the regulation of biofilm formation in S. aureus. Conclusion These findings demonstrate that AI-2 can decrease biofilm formation in S. aureus via an icaR-activation pathway. This study may provide clues for therapy in S. aureus biofilm-associated infection.

  17. Inactivation of ferric uptake regulator (Fur) attenuates Helicobacter pylori J99 motility by disturbing the flagellar motor switch and autoinducer-2 production.

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    Lee, Ai-Yun; Kao, Cheng-Yen; Wang, Yao-Kuan; Lin, Ssu-Yuan; Lai, Tze-Ying; Sheu, Bor-Shyang; Lo, Chien-Jung; Wu, Jiunn-Jong

    2017-08-01

    Flagellar motility of Helicobacter pylori has been shown to be important for the bacteria to establish initial colonization. The ferric uptake regulator (Fur) is a global regulator that has been identified in H. pylori which is involved in the processes of iron uptake and establishing colonization. However, the role of Fur in H. pylori motility is still unclear. Motility of the wild-type, fur mutant, and fur revertant J99 were determined by a soft-agar motility assay and direct video observation. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. Single bacterial motility and flagellar switching were observed by phase-contrast microscopy. Autoinducer-2 (AI-2) production in bacterial culture supernatant was analyzed by a bioluminescence assay. The fur mutant showed impaired motility in the soft-agar assay compared with the wild-type J99 and fur revertant. The numbers and lengths of flagellar filaments on the fur mutant cells were similar to those of the wild-type and revertant cells. Phenotypic characterization showed similar swimming speed but reduction in switching rate in the fur mutant. The AI-2 production of the fur mutant was dramatically reduced compared with wild-type J99 in log-phase culture medium. These results indicate that Fur positively modulates H. pylori J99 motility through interfering with bacterial flagellar switching. © 2017 John Wiley & Sons Ltd.

  18. New Insights into Autoinducer-2 Signaling as a Virulence Regulator in a Mouse Model of Pneumonic Plague

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    Fitts, Eric C.; Andersson, Jourdan A.; Kirtley, Michelle L.; Sha, Jian; Erova, Tatiana E.; Chauhan, Sadhana; Motin, Vladimir L.

    2016-01-01

    ABSTRACT The Enterobacteriaceae family members, including the infamous Yersinia pestis, the causative agent of plague, have a highly conserved interbacterial signaling system that is mediated by the autoinducer-2 (AI-2) quorum-sensing molecule. The AI-2 system is implicated in regulating various bacterial virulence genes in diverse environmental niches. Deletion of the gene encoding the synthetic enzyme for the AI-2 substrate, luxS, leads to either no significant change or, paradoxically, an increase in in vivo bacterial virulence. We showed that deletion of the rbsA and lsrA genes, components of ABC transport systems that interact with AI-2, synergistically disrupted AI-2 signaling patterns and resulted in a more-than-50-fold decrease in Y. pestis strain CO92 virulence in a stringent pneumonic plague mouse model. Deletion of luxS or lsrK (encoding AI-2 kinase) from the ΔrbsA ΔlsrA background strain or complementation of the ΔrbsA ΔlsrA mutant with the corresponding gene(s) reverted the virulence phenotype to that of the wild-type Y. pestis CO92. Furthermore, the administration of synthetic AI-2 in mice infected with the ΔrbsA ΔlsrA ΔluxS mutant strain attenuated this triple mutant to a virulence phenotype similar to that of the ΔrbsA ΔlsrA strain in a pneumonic plague model. Conversely, the administration of AI-2 to mice infected with the ΔrbsA ΔlsrA ΔluxS ΔlsrK mutant did not rescue animals from lethality, indicating the importance of the AI-2–LsrK axis in regulating bacterial virulence. By performing high-throughput RNA sequencing, the potential role of some AI-2-signaling-regulated genes that modulated bacterial virulence was determined. We anticipate that the characterization of AI-2 signaling in Y. pestis will lead to reexamination of AI-2 systems in other pathogens and that AI-2 signaling may represent a broad-spectrum therapeutic target to combat antibiotic-resistant bacteria, which represent a global crisis of the 21st century. IMPORTANCE

  19. Streptococcus gordonii LuxS/autoinducer-2 quorum-sensing system modulates the dual-species biofilm formation with Streptococcus mutans.

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    Wang, Xiao; Li, Xiaolan; Ling, Junqi

    2017-07-01

    Dental plaques are mixed-species biofilms that are related to the development of dental caries. Streptococcus mutans (S. mutans) is an important cariogenic bacterium that forms mixed-species biofilms with Streptococcus gordonii (S. gordonii), an early colonizer of the tooth surface. The LuxS/autoinducer-2(AI-2) quorum sensing system is involved in the regulation of mixed-species biofilms, and AI-2 is proposed as a universal signal for the interaction between bacterial species. In this work, a S. gordonii luxS deficient strain was constructed to investigate the effect of the S. gordonii luxS gene on dual-species biofilm formed by S. mutans and S. gordonii. In addition, AI-2 was synthesized in vitro by incubating recombinant LuxS and Pfs enzymes of S. gordonii together. The effect of AI-2 on S. mutans single-species biofilm formation and cariogenic virulence gene expression were also assessed. The results showed that luxS disruption in S. gordonii altered dual-species biofilm formation, architecture, and composition, as well as the susceptibility to chlorhexidine. And the in vitro synthesized AI-2 had a concentration-dependent effect on S. mutans biofilm formation and virulence gene expression. These findings indicate that LuxS/AI-2 quorum-sensing system of S. gordonii plays a role in regulating the dual-species biofilm formation with S. mutans. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Autoinducer-2 Quorum Sensing Influences Viability of Escherichia coli O157:H7 under Osmotic and In Vitro Gastrointestinal Stress Conditions

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    Hyunjoon Park

    2017-06-01

    Full Text Available Bacteria use autoinducer molecules to communicate both at intra-species and inter-species levels by quorum sensing. One such cell density-dependent signaling system is the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2. Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy toward designing of new antimicrobial agents. However, recent studies indicate that the AI-2 system is also involved in adaptation and survival under environmental stress conditions. Therefore, we hypothesized that interaction between quorum sensing and environmental conditions may be critical in influencing predicted results in a control and when combating of target pathogens. We investigated the growth of enterohemorrhagic Escherichia coli O157:H7 (EHEC and its luxS-deficient (non AI-2 producing mutant strain under various stress conditions, and found significant differences in the growth rate under osmotic stress. Moreover, we could also show the impact of the AI-2 molecule on viability in the gastrointestinal tract model representing a complex environmental condition. Differences in vital responses of the strains suggest that AI-2 quorum sensing has a significant influence on the viability of EHEC under environmental stress conditions.

  1. Autoinducer-2 Quorum Sensing Influences Viability ofEscherichia coliO157:H7 under Osmotic andIn VitroGastrointestinal Stress Conditions.

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    Park, Hyunjoon; Lee, Kyuyeon; Yeo, Soyoung; Shin, Heuynkil; Holzapfel, Wilhelm H

    2017-01-01

    Bacteria use autoinducer molecules to communicate both at intra-species and inter-species levels by quorum sensing. One such cell density-dependent signaling system is the luxS -mediated universal quorum sensing using autoinducer-2 (AI-2). Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy toward designing of new antimicrobial agents. However, recent studies indicate that the AI-2 system is also involved in adaptation and survival under environmental stress conditions. Therefore, we hypothesized that interaction between quorum sensing and environmental conditions may be critical in influencing predicted results in a control and when combating of target pathogens. We investigated the growth of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and its luxS -deficient (non AI-2 producing) mutant strain under various stress conditions, and found significant differences in the growth rate under osmotic stress. Moreover, we could also show the impact of the AI-2 molecule on viability in the gastrointestinal tract model representing a complex environmental condition. Differences in vital responses of the strains suggest that AI-2 quorum sensing has a significant influence on the viability of EHEC under environmental stress conditions.

  2. Autoinducer-2 of Streptococcus mitis as a target molecule to inhibit pathogenic multi-species biofilm formation in vitro and in an endotracheal intubation rat model

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    Zhengli eWang

    2016-02-01

    Full Text Available AbstractStreptococcus mitis (S. mitis and Pseudomonas aeruginosa (P. aeruginosa are typically found in the upper respiratory tract of infants. We previously found that P. aeruginosa and S. mitis were two of the most common bacteria in biofilms on newborns’ endotracheal tubes (ETTs and in their sputa and that S. mitis was able to produce autoinducer-2 (AI-2, but P. aeruginosa was not. Recently, we also found that exogenous AI-2 and S. mitis could influence the behaviors of P. aeruginosa. We hypothesized that S. mitis contributes to this interspecies interaction and that inhibition of AI-2 could result in inhibition of these effects. To test this hypothesis, we selected PAO1 as a representative model strain of P. aeruginosa and evaluated the effect of S. mitis as well as an AI-2 analog (D-ribose on mono- and co-culture biofilms in both in vitro and in vivo models. In this context, S. mitis promoted PAO1 biofilm formation and pathogenicity. Dual-species (PAO1 and S. mitis biofilms exhibited higher expression of quorum sensing genes than single-species (PAO1 biofilms did. Additionally, ETTs covered in dual-species biofilms increased the mortality rate and aggravated lung infection compared with ETTs covered in mono-species biofilms in an endotracheal intubation rat model, all of which was inhibited by D-ribose. Our results demonstrated that S. mitis AI-2 plays an important role in interspecies interactions with PAO1 and may be a target for inhibition of biofilm formation and infection in ventilator-associated pneumonia.

  3. Exploring novel food proteins and processing technologies

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine

    2016-01-01

    Foods rich in protein are nowadays high in demand worldwide. To ensure a sustainable supply and a high quality of protein foods, novel food proteins and processing technologies need to be explored to understand whether they can be used for the development of high-quality protein foods. Therefore,

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

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

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

  5. 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…

  6. The impact of processing on amino acid racemization and protein quality in processed animal proteins of poultry origin

    OpenAIRE

    Federica Bellagamba; Fabio Caprino; Tiziana Mentasti; Mauro Vasconi; Vittorio Maria Moretti

    2015-01-01

    Re-authorization of processed animal proteins (PAPs) in EU, derived from by-products of human food production, could increase manufacturing of proteins for feed ingredients and reduce the need of imported proteins mainly of plant origin. The PAPs production is largely done by the rendering process during which authorized animal by-products are heat treated to extract valuable protein and animal fat, ensuring sterilizing conditions of raw incoming materials. Proteins exposure to certain proces...

  7. Of proteins and processing: mechanisms of protein damage upon rapeseed processing and their effects on nutritional value

    NARCIS (Netherlands)

    Salazar Villanea, Sergio

    2017-01-01

    Hydrothermal processing is a common practice during the manufacture of protein-rich feed ingredients, such as rapeseed meal (RSM), and feeds. This processing step can induce physical and chemical changes to the proteins, thereby reducing the digestibility and utilization of crude protein (CP) and

  8. Of proteins and processing: mechanisms of protein damage upon rapeseed processing and their effects on nutritional value

    NARCIS (Netherlands)

    Salazar Villanea, Sergio

    2017-01-01

    Hydrothermal processing is a common practice during the manufacture of protein-rich feed ingredients, such as rapeseed meal (RSM), and feeds. This processing step can induce physical and chemical changes to the proteins, thereby reducing the digestibility and utilization of crude protein (CP) and

  9. Retinoblastoma protein: a central processing unit

    Indian Academy of Sciences (India)

    Prakash

    most important mechanism of cell cycle arrest caused by the. Rb protein. How exactly does the Rb–E2F complex actively repress transcription once it is anchored to E2F-regulated promoters? One important mechanism of gene transcription regulation is through modification of chromatin structure, mainly by histone ...

  10. Retinoblastoma protein: a central processing unit

    Indian Academy of Sciences (India)

    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 ...

  11. Assessment of protein quality of processed melon seed as a ...

    African Journals Online (AJOL)

    Ninety six day - old broiler chicks were used in a 4 - week feeding trial to determine the protein quality of processed melon seed as a component of broiler chick diet. The protein quality was assessed using blood associated parameters including Haemoglobin indices and selected serum enzymes. The processed melon ...

  12. Retinoblastoma protein: a central processing unit

    Indian Academy of Sciences (India)

    Prakash

    G1-to-S transition (Goodrich et al. 1991; Hinds et al. 1992). Cyclin-dependent kinases ... This protein regulates critical G1-to-S phase transition through interaction with the. E2F family of cell-cycle transcription factors ..... He S, Cook B L, Deverman B E, Weihe U, Zhang F, Prachand V,. Zheng J and Weintraub S J 2000 E2F is ...

  13. Protein substitution to produce a processed cheese with high ...

    African Journals Online (AJOL)

    Hoida A.M. El-Shazly

    KEYWORDS. Processed cheese;. Branched chain amino acids;. Ras cheese;. Kariesh cheese;. Protein replacement;. Chemical and sensory analysis;. Liver;. Brain; ... Effect of 2.5%, 5% and 10% protein-replacement cheese formulas was evaluated among .... and Robbins [22] using HPLC equipment Wallac fluorometer.

  14. The impact of processing on amino acid racemization and protein quality in processed animal proteins of poultry origin

    Directory of Open Access Journals (Sweden)

    Federica Bellagamba

    2015-06-01

    Full Text Available Re-authorization of processed animal proteins (PAPs in EU, derived from by-products of human food production, could increase manufacturing of proteins for feed ingredients and reduce the need of imported proteins mainly of plant origin. The PAPs production is largely done by the rendering process during which authorized animal by-products are heat treated to extract valuable protein and animal fat, ensuring sterilizing conditions of raw incoming materials. Proteins exposure to certain processing conditions induces two important chemical changes, racemization of amino acids and formation of cross-linked amino acid. These changes are associated with appreciable reduction of protein digestibility and nutritional value. The aim of this study was to verify the effect of heat treatment on amino acid racemization in processed animal proteins of poultry origin and related nutritional implications by evaluation of their in vitro digestibility. The results reported confirm the detection of racemized amino acids in processed animal proteins, especially D-aspartic acid, as realistic indicators of thermal treatments during PAP manufacturing. In our results, the severe (115°C and prolonged heat treatment (180 minutes revealed a D-Asp content of 28.1%. Prolongation of temperature treatment (20, 30 and 180 min, at 115°C significantly (P<0.05 affects in vitro protein digestibility, which decrease from 86.0%, in no-treated sample, to 78.3% and 79.1% after 20 and 30 min, respectively, and to 76.3% after 180 min. The processing conditions applied during PAPs preparations and the racemization of proteins amino acids may reasonably be involved in the loss of protein quality.

  15. Protein import into plant mitochondria: signals, machinery, processing, and regulation.

    Science.gov (United States)

    Murcha, Monika W; Kmiec, Beata; Kubiszewski-Jakubiak, Szymon; Teixeira, Pedro F; Glaser, Elzbieta; Whelan, James

    2014-12-01

    The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Chloroplast Protein Turnover: The Influence of Extraplastidic Processes, Including Autophagy

    Directory of Open Access Journals (Sweden)

    Masanori Izumi

    2018-03-01

    Full Text Available Most assimilated nutrients in the leaves of land plants are stored in chloroplasts as photosynthetic proteins, where they mediate CO2 assimilation during growth. During senescence or under suboptimal conditions, chloroplast proteins are degraded, and the amino acids released during this process are used to produce young tissues, seeds, or respiratory energy. Protein degradation machineries contribute to the quality control of chloroplasts by removing damaged proteins caused by excess energy from sunlight. Whereas previous studies revealed that chloroplasts contain several types of intraplastidic proteases that likely derived from an endosymbiosed prokaryotic ancestor of chloroplasts, recent reports have demonstrated that multiple extraplastidic pathways also contribute to chloroplast protein turnover in response to specific cues. One such pathway is autophagy, an evolutionarily conserved process that leads to the vacuolar or lysosomal degradation of cytoplasmic components in eukaryotic cells. Here, we describe and contrast the extraplastidic pathways that degrade chloroplasts. This review shows that diverse pathways participate in chloroplast turnover during sugar starvation, senescence, and oxidative stress. Elucidating the mechanisms that regulate these pathways will help decipher the relationship among the diverse pathways mediating chloroplast protein turnover.

  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. 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.

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

    Science.gov (United States)

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

    2013-06-01

    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 expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Isolation and processing of silk proteins for biomedical applications.

    Science.gov (United States)

    Kundu, Banani; Kurland, Nicholas E; Yadavalli, Vamsi K; Kundu, Subhas C

    2014-09-01

    Silk proteins of silkworms are chiefly composed of core fibroin protein and glycoprotein sericin that glues fibroin. Unique mechanical properties, cyto-compatibility and controllable biodegradability facilitate the use of fibroin in biomedical applications. Sericin serves as additive in cosmetic and food industries, as mitotic factor in cell culture media, anti-cancerous drug, anticoagulant and as biocompatible coating. For all these uses; aqueous solutions of silk proteins are preferred. Therefore, an accurate understanding of extraction procedure of silk proteins from their sources is critical. A number of protocols exist, amongst which it is required to settle a precise and easy one with desired yield and least down-stream processing. Here, we report extraction of proteins employing methods mentioned in literature using cocoons of mulberry and nonmulberry silks. This study reveals sodium carbonate salt-boiling system is the most efficient sericin extraction procedure for all silk variants. Lithium bromide is observed as the effective fibroin dissolution system for mulberry silk cocoons; whereas heterogeneous species-dependent result is obtained in case of nonmulberry species. We further show the effect of common post processing on nanoscale morphology of mulberry silk fibroin films. This knowledge eases the adoption and fabrication of silk biomaterials in devices and therapeutic delivery systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Proteolytic events in the processing of secreted proteins in fungi.

    Science.gov (United States)

    Calmels, T P; Martin, F; Durand, H; Tiraby, G

    1991-01-01

    Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

  2. Process considerations for protein engineering of ω-Transaminase

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson; Schwarze, Daniel; Tufvesson, Pär

    . However, often wild type enzyme does not fit the requirements of the process conditions, where high substrate and product concentrations as well as high productivity demands of the catalyst (g product per g biocatalyst), are key to economic feasibility. The question thus arises whether to fit the process...... 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. It was seen...

  3. 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.

  4. MUFOLD-DB: a processed protein structure database for protein structure prediction and analysis.

    Science.gov (United States)

    He, Zhiquan; Zhang, Chao; Xu, Yang; Zeng, Shuai; Zhang, Jingfen; Xu, Dong

    2014-01-01

    Protein structure data in Protein Data Bank (PDB) are widely used in studies of protein function and evolution and in protein structure prediction. However, there are two main barriers in large-scale usage of PDB data: 1) PDB data are highly redundant in terms of sequence and structure similarity; and 2) many PDB files have issues due to inconsistency of data and standards as well as missing residues, so that automated retrieval and analysis are often difficult. To address these issues, we have created MUFOLD-DB http://mufold.org/mufolddb.php, a web-based database, to collect and process the weekly PDB files thereby providing users with non-redundant, cleaned and partially-predicted structure data. For each of the non-redundant sequences, we annotate the SCOP domain classification and predict structures of missing regions by loop modelling. In addition, evolutional information, secondary structure, disorder region, and processed three-dimensional structure are computed and visualized to help users better understand the protein. MUFOLD-DB integrates processed PDB sequence and structure data and multiple computational results, provides a friendly interface for users to retrieve, browse and download these data, and offers several useful functionalities to facilitate users' data operation.

  5. Processing of precursor proteins by preparations of oviduct microsomes.

    Science.gov (United States)

    Thibodeau, S N; Walsh, K A

    1980-01-01

    The signal peptidases from both chicken oviduct and dog pancreas microsomes have been detected by a post-translational assay and, in the case of dog microsomes, by a cotranslational assay. This proteolytic activity is not sensitive to a variety of conventional protease inhibitors under the conditions used in these assays. Using a synthetic ester substrate, two additional activities have been observed in oviduct microsomes, but neither appears to correspond to the signal peptidase activity. Using cDNA as a probe for specific mRNAs, it was shown that polysomes, in the process of synthesizing secretory proteins, bind to microsomal membrane vesicles. This binding appears to be dependent on the nature of the translation product (presumably the specific signal peptide) rather than on the mRNA itself. In addition, pretreatment of the membrane vesicles with N-ethyl maleimide prevents the vectorial discharge and processing of several secretory proteins but does not inhibit the binding of their polysomes to membranes.

  6. Rank Protein Immunolabeling during Bone-Implant Interface Healing Process

    OpenAIRE

    ?vila Souza, Francisley; Pereira Queiroz, Thallita; Rodrigues Luvizuto, Elo?; Nishioka, Renato Sussumu; Garcia-JR, Idelmo Rangel; de Carvalho, Paulo S?rgio Perri; Okamoto, Roberta

    2010-01-01

    The purpose of this paper was to evaluate the expression of RANK protein during bone-healing process around machined surface implants. Twenty male Wistar rats, 90 days old, after having had a 2?mm diameter and 6 mm long implant inserted in their right tibias, were evaluated at 7, 14, 21, and 42 days after healing. After obtaining the histological samples, slides were subjected to RANK immunostaining reaction. Results were quantitatively evaluated. Results. Immunolabeling analysis showed expre...

  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

  8. Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia.

    Science.gov (United States)

    Buhimschi, Irina A; Nayeri, Unzila A; Zhao, Guomao; Shook, Lydia L; Pensalfini, Anna; Funai, Edmund F; Bernstein, Ira M; Glabe, Charles G; Buhimschi, Catalin S

    2014-07-16

    Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state-dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state-dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer's β-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the β-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of β-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia. Copyright © 2014, American Association for the Advancement of Science.

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

    International Nuclear Information System (INIS)

    El-Niely, H.F.G

    2007-01-01

    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

  10. Rank Protein Immunolabeling during Bone-Implant Interface Healing Process

    Directory of Open Access Journals (Sweden)

    Francisley Ávila Souza

    2010-01-01

    Full Text Available The purpose of this paper was to evaluate the expression of RANK protein during bone-healing process around machined surface implants. Twenty male Wistar rats, 90 days old, after having had a 2 mm diameter and 6 mm long implant inserted in their right tibias, were evaluated at 7, 14, 21, and 42 days after healing. After obtaining the histological samples, slides were subjected to RANK immunostaining reaction. Results were quantitatively evaluated. Results. Immunolabeling analysis showed expressions of RANK in osteoclast and osteoblast lineage cells. The statistical analysis showed an increase in the expression of RANK in osteoblasts at 7 postoperative days and a gradual decrease during the chronology of the healing process demonstrated by mild cellular activity in the final stage (P<.05. Conclusion. RANK immunolabeling was observed especially in osteoclast and osteoblast cells in primary bone during the initial periods of bone-healing/implant interface.

  11. Rank Protein Immunolabeling during Bone-Implant Interface Healing Process

    Science.gov (United States)

    Ávila Souza, Francisley; Pereira Queiroz, Thallita; Rodrigues Luvizuto, Eloá; Nishioka, Renato Sussumu; Garcia-JR, Idelmo Rangel; de Carvalho, Paulo Sérgio Perri; Okamoto, Roberta

    2010-01-01

    The purpose of this paper was to evaluate the expression of RANK protein during bone-healing process around machined surface implants. Twenty male Wistar rats, 90 days old, after having had a 2 mm diameter and 6 mm long implant inserted in their right tibias, were evaluated at 7, 14, 21, and 42 days after healing. After obtaining the histological samples, slides were subjected to RANK immunostaining reaction. Results were quantitatively evaluated. Results. Immunolabeling analysis showed expressions of RANK in osteoclast and osteoblast lineage cells. The statistical analysis showed an increase in the expression of RANK in osteoblasts at 7 postoperative days and a gradual decrease during the chronology of the healing process demonstrated by mild cellular activity in the final stage (P < .05). Conclusion. RANK immunolabeling was observed especially in osteoclast and osteoblast cells in primary bone during the initial periods of bone-healing/implant interface. PMID:20706673

  12. Process development of a FGF21 protein-antibody conjugate.

    Science.gov (United States)

    Dirksen, Anouk; Davis, Keith A; Collins, Joe T; Bhattacharya, Keshab; Finneman, Jari I; Pepin, Erin L; Ryczek, Jeffrey S; Brown, Paul W; Wellborn, William B; Mangalathillam, Ratish; Evans, Brad P; Pozzo, Mark J; Finn, Rory F

    2017-09-26

    A scalable, viable process was developed for the Fibroblast Growth Factor 21 (FGF21) protein-antibody conjugate, CVX-343, an extended half-life therapeutic for the treatment of metabolic disease. CVX-343 utilizes the CovX antibody scaffold technology platform that was specifically developed for peptide and protein half-life extension. CVX-343 is representative of a growing number of complex novel peptide- and protein-based bioconjugate molecules currently being explored as therapeutic candidates. The complexity of these bioconjugates, assembled using well-established chemistries, can lead to very difficult production schemes requiring multiple starting materials and a combination of diverse technologies. Key improvements had to be made to the original CVX-343 Phase 1 manufacturing process in preparation for Phase 3 and commercial manufacturing. A strategy of minimizing FGF21 A129C dimerization and stabilizing the FGF21 A129C Drug Substance Intermediate (DSI), linker, and activated FGF21 intermediate was pursued. The use of tris(2-carboxyethyl)phosphine (TCEP) to prevent FGF21 A129C dimerization through disulfide formation was eliminated. FGF21 A129C dimerization and linker hydrolysis were minimized by formulating and activating FGF21 A129C at acidic instead of neutral pH. An activation use test was utilized to guide FGF21 A129C pooling in order to minimize misfolds, dimers, and misfolded dimers in the FGF21 A129C DSI. After final optimization of reaction conditions, a process was established that reduced the consumption of FGF21 A129C by 36% (from 4.7 to 3.0 equivalents) and the consumption of linker by 55% (from 1.4 to 0.95 equivalents for a smaller required amount of FGF21 A129C ). The overall process time was reduced from ∼5 to ∼3 days. The product distribution improved from containing ∼60% to ∼75% desired bifunctionalized (+2 FGF21) FGF21-antibody conjugate in the crude conjugation mixture and from ∼80% to ∼85% in the final CVX-343 Drug Substance

  13. Cyanobacteria gene and protein sequences in diurnal oscillation metabolic processes

    Science.gov (United States)

    Tremberger, George, Jr.; Holden, T.; Cheung, E.; Dehipawala, S.; Gadura, N.; Golebiewska, U.; Valentin, K.; Smulczeski, M.; Satizabal, W.; Schneider, P.; Lieberman, D.; Cheung, T.

    2010-09-01

    Daytime photosynthesis and nighttime nitrogen fixation metabolic processes have been reported in the bacterium, Cyanothece 51142. The organism's auto-fluorescence with 532 nm excitation would place cyanobacteria at the forefront in the remote sensing of microbial activity in astrobiology. The sensitivity of nitrogenase to oxygen was studied in terms of sequence nucleotide fluctuation. A nucleotide sequence fractal dimension can be calculated from a numerical series consisting of the atomic numbers of each nucleotide. The fractal dimension and Shannon entropy form a two-dimensional measure that is useful in assessing evolutionary pressures. The studied sequences include nitrogenase iron protein NifH, nitrogenase molybdenum-iron protein alpha chain NifD and beta chain NifK. The photosynthesis-lacking UCYN-A cyanobacterium as reported recently in the journal, Nature, was observed to have the lowest entropy with relatively high fractal dimension values in the studied NifH, NifD and NifH sequences. The fractal dimension of NifH sequences correlates with the NifD sequence values with an R-square of 0.91 (N = 8). The Shannon mononucleotide entropy of NifD sequences correlates with the NifK sequence values with an R-square value of 0.92 (N = 8). The observed strong correlation suggests the presence of gradual evolutionary pressure among the studied cyanobacteria, and throws light on the reported paradox in evolution for the case of UCYN-A. The results show that diurnal oscillation metabolic processes in cyanobacteria (including the photosynthesis-deficient case) are not associated with extraordinary evolutionary pressures and thus are processes consistent with putative astrobiological organisms.

  14. Distinct amyloid precursor protein processing machineries of the olfactory system.

    Science.gov (United States)

    Kim, Jae Yeon; Rasheed, Ameer; Yoo, Seung-Jun; Kim, So Yeun; Cho, Bongki; Son, Gowoon; Yu, Seong-Woon; Chang, Keun-A; Suh, Yoo-Hun; Moon, Cheil

    2018-01-01

    Processing of amyloid precursor protein (APP) occurs through sequential cleavages first by β-secretase and then by the γ-secretase complex. However, abnormal processing of APP leads to excessive production of β-amyloid (Aβ) in the central nervous system (CNS), an event which is regarded as a primary cause of Alzheimer's disease (AD). In particular, gene mutations of the γ-secretase complex-which contains presenilin 1 or 2 as the catalytic core-could trigger marked Aβ accumulation. Olfactory dysfunction usually occurs before the onset of typical AD-related symptoms (eg, memory loss or muscle retardation), suggesting that the olfactory system may be one of the most vulnerable regions to AD. To date however, little is known about why the olfactory system is affected so early by AD prior to other regions. Thus, we examined the distribution of secretases and levels of APP processing in the olfactory system under either healthy or pathological conditions. Here, we show that the olfactory system has distinct APP processing machineries. In particular, we identified higher expressions levels and activity of γ-secretase in the olfactory epithelium (OE) than other regions of the brain. Moreover, APP c-terminal fragments (CTF) are markedly detected. During AD progression, we note increased expression of presenilin2 of γ-secretases in the OE, not in the OB, and show that neurotoxic Aβ*56 accumulates more quickly in the OE. Taken together, these results suggest that the olfactory system has distinct APP processing machineries under healthy and pathological conditions. This finding may provide a crucial understanding of the unique APP-processing mechanisms in the olfactory system, and further highlights the correlation between olfactory deficits and AD symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. A general sequence processing and analysis program for protein engineering.

    Science.gov (United States)

    Stafford, Ryan L; Zimmerman, Erik S; Hallam, Trevor J; Sato, Aaron K

    2014-10-27

    Protein engineering projects often amass numerous raw DNA sequences, but no readily available software combines sequence processing and activity correlation required for efficient lead identification. XLibraryDisplay is an open source program integrated into Microsoft Excel for Windows that automates batch sequence processing via a simple step-by-step, menu-driven graphical user interface. XLibraryDisplay accepts any DNA template which is used as a basis for trimming, filtering, translating, and aligning hundreds to thousands of sequences (raw, FASTA, or Phred PHD file formats). Key steps for library characterization through lead discovery are available including library composition analysis, filtering by experimental data, graphing and correlating to experimental data, alignment to structural data extracted from PDB files, and generation of PyMOL visualization scripts. Though larger data sets can be handled, the program is best suited for analyzing approximately 10 000 or fewer leads or naïve clones which have been characterized using Sanger sequencing and other experimental approaches. XLibraryDisplay can be downloaded for free from sourceforge.net/projects/xlibrarydisplay/ .

  16. 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.

  17. Protein substitution to produce a processed cheese with high ...

    African Journals Online (AJOL)

    Multiple studies report the beneficial effects of BCAAs supplementation to improve plasma amino acids imbalance, several neurologic diseases, protein energy malnutrition, and subsequently the survival rate of cirrhotic patients. Methods: In the present study we used a protein substitution technique to synthesize a new ...

  18. Effective strategies for host cell protein clearance in downstream processing of monoclonal antibodies and Fc-fusion proteins.

    Science.gov (United States)

    Li, Yifeng

    2017-06-01

    Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product. Hence reducing them to the generally accepted level can be challenging. This article reviews effective HCP removing strategies at different stages of downstream process for monoclonal antibodies and Fc-fusion proteins. When used in combination, these strategies can greatly enhance the chance of meeting the drug substance specifications for residual HCP. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Disentangling interfacial redox processes of proteins by SERR spectroscopy.

    Science.gov (United States)

    Murgida, Daniel H; Hildebrandt, Peter

    2008-05-01

    Surface-enhanced resonance-Raman spectroelectrochemistry represents a powerful approach for studying the structure and reaction dynamics of redox proteins immobilized on biocompatible electrodes in fundamental and applied sciences. Using this approach it has been recently shown that electric fields of biologically relevant magnitude are able to influence crucial parameters for the functioning of a variety of soluble and membrane bound heme proteins. Electric field effects discussed in this tutorial review include modulation of redox potentials, reorganization energies, protein dynamics and redox-linked structural changes.

  20. Effect of four processed animal proteins in the diet on digestibility and performance in laying hens.

    NARCIS (Netherlands)

    Krimpen, van M.M.; Veldkamp, T.; Binnendijk, G.P.; Veer, de R.

    2010-01-01

    An experiment was performed to investigate the effect of animal vs. vegetable protein sources in the diet of laying hens on the development of hen performance. A diet containing protein sources of only vegetable origin was compared with 4 diets, each containing 1 of 4 processed animal proteins

  1. Effect of four processed animal proteins in the diet on behavior in laying hens

    NARCIS (Netherlands)

    Krimpen, van M.M.; Veldkamp, T.; Binnendijk, G.P.; Veer, de R.

    2011-01-01

    An experiment was performed to investigate the effect of animal versus vegetable protein sources in the diet on the development of behavior in laying hens. A diet containing protein sources of only vegetable origin was compared with four diets, each containing one of four processed animal proteins

  2. 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).

  3. 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

  4. Improving protein stabilization by spray drying : formulation and process development

    NARCIS (Netherlands)

    Grasmeijer, Niels

    2016-01-01

    There is an increasing interest for dried protein formulations in pharmacy. They may offer several advantages over aqueous formulations, such as ease of storage, longer shelf life, and use for solid dosage forms. However, the mechanisms underlying stabilization in the solid state are not yet fully

  5. 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.

  6. 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.

  7. 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

  8. 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. © 2016 The Authors.

  9. 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.

  10. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    Science.gov (United States)

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  11. Monocyte chemoattractant protein-1: a key mediator in inflammatory processes.

    Science.gov (United States)

    Melgarejo, Esther; Medina, Miguel Angel; Sánchez-Jiménez, Francisca; Urdiales, José Luis

    2009-05-01

    Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant for monocytes and macrophages to areas of inflammation. MCP-1 is a prototypical chemokine subject to coordinated regulation by immunomodulatory agents. Since MCP-1 is implicated in multiple inflammatory diseases, it is a potential target for the treatment of these disorders. In this review, we will provide background information and summarize the MCP-1 structure and signaling pathways. Its involvement in multiple diseases, such as tumour development, atherogenesis and rare autoimmune diseases is also revised.

  12. 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.

  13. In vitro bioaccessibility of proteins and lipids of pH-shift processed Nannochloropsis oculata microalga.

    Science.gov (United States)

    Cavonius, L R; Albers, E; Undeland, I

    2016-04-01

    The pH-shift process fractionates biomass into soluble proteins and insoluble fractions, followed by precipitation and recovery of the solubilized proteins. Nannochloropsis oculata in seawater was subjected to the pH-shift process, followed by digestion of various intermediates and product fractions of the process, using the Infogest in vitro digestion model (Minekus et al., 2014) with added gastric lipase. As measures for protein and lipid accessibility, degrees of protein hydrolysis and fatty acid liberation were assessed post-digestion and compared to the amounts of peptide bonds and total fatty acids present in the raw materials. Results showed that neither proteins nor lipids of intact Nannochloropsis cells were accessible to the mammalian digestive enzymes used in the digestion model. Cell disruption, and to a lesser extent, further pH-shift processing with protein solubilisation at pH 7 or pH 10, increased the accessibility of lipids. For proteins, differences amongst the pH-shift processed materials were non-significant, though pre-freezing the product prior to digestion increased the accessibility from 32% to 47%. For fatty acids, pH-shift process-products gave rise to 43% to 52% lipolysis, with higher lipolysis for products solubilised at pH 10 as opposed to pH 7. Our results indicate the importance of processing to produce an algal product that has beneficial nutritional properties when applied as food or feed.

  14. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process.

    Science.gov (United States)

    Singh, Anupam; Upadhyay, Vaibhav; Upadhyay, Arun Kumar; Singh, Surinder Mohan; Panda, Amulya Kumar

    2015-03-25

    Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

  15. Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein.

    Science.gov (United States)

    Yeh, S D; Gonsalves, D

    1985-05-01

    The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.

  16. Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation.

    Science.gov (United States)

    Lott, Kaylen; Li, Jun; Fisk, John C; Wang, Hao; Aletta, John M; Qu, Jun; Read, Laurie K

    2013-10-08

    Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of Trypanosoma brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. T. brucei is a protozoan parasite that causes lethal African sleeping sickness in humans and nagana in livestock, thereby imposing a significant medical and economic burden on sub-Saharan Africa. The parasite encounters very different environments as it cycles between mammalian and insect hosts, and must exert cellular responses to these varying milieus. One mechanism by which all cells respond to changing

  17. 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

  18. hPDB – Haskell library for processing atomic biomolecular structures in protein data bank format

    OpenAIRE

    Gajda, Michał Jan

    2013-01-01

    Background Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. Findings I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The propo...

  19. Enhancement of Protein and Pigment Content in Two Chlorella Species Cultivated on Industrial Process Water

    DEFF Research Database (Denmark)

    Safafar, Hamed; Uldall Nørregaard, Patrick; Ljubic, Anita

    2016-01-01

    composition of biomass. Variations in proteins, lipid, fatty acid composition, amino acids, tocopherols, and pigments were studied. Both species grew well in industrial process water. The contents of proteins were affected significantly by the growth media and cultivation duration. Microalga Chlorella...... is an environmentally friendly, sustainable bioremediation method with added value biomass production and resource valorization, since the resulting biomass also presented a good source of proteins, amino acids, and carotenoids for potential use in aquaculture feed industry....

  20. 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

  1. Effect of primary emulsions on microsphere size and protein-loading in the double emulsion process.

    Science.gov (United States)

    Maa, Y F; Hsu, C C

    1997-01-01

    Incorporation of a protein drug in microspheres made of a hydrophobic polymer is commonly achieved via double liquid-liquid emulsification (w/o/w) or by dispersing a powdered protein in a polymer solution followed by liquid-liquid emulsification (s/o/w). This study focused on the effect of the first operating step in both processes on the size and protein-loading of the microspheres. Bovine serum albumin (BSA) was used as the model protein and poly(methyl methacrylate) (PMMA) was used as the model polymer. The w/o emulsion was characterized based on the degree of emulsion fineness which was controlled using rotor/stator homogenization. The s/o emulsion was characterized based on protein powder size and shape. Protein powders of different sizes and shapes were produced using different powder preparation methods. In both emulsification processes, the second operating step which produced the microspheres was conducted in either a continuously stirred tank reactor (CSTR) or a static mixer. The size of the microspheres thus prepared was found to increase with increasing size of the protein powder in the s/o/w system but increase with decreasing size of the liquid emulsion droplets in the w/o/w system. Empirical correlations can accurately predict the size of the microspheres if the size of w/o emulsion droplets and protein powder is 10 x less than the microsphere size. Protein loading in the microspheres decreased with respect to increases in w/o emulsion droplet size or in protein powder size. We propose that these phenomena are attributed to two mechanisms, fragmentation along the weak routes in the w/o/w system and particle redistribution as the result of terminal velocity in the s/o/w system. The role of protein powder shape was not significant until the protein powder size exceeded 5 microns. Irregular-shaped protein powders resulted in lower encapsulation efficiency than spherical-shaped protein powders.

  2. Effect of processing intensity on immunologically active bovine milk serum proteins

    NARCIS (Netherlands)

    Brick, Tabea; Ege, Markus; Boeren, Sjef; Böck, Andreas; Mutius, Von Erika; Vervoort, Jacques; Hettinga, Kasper

    2017-01-01

    Consumption of raw cow’s milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general

  3. Proteomic study on the stability of proteins in bovine, camel, and caprine milk sera after processing

    NARCIS (Netherlands)

    Zhang, Lina; Boeren, Sjef; Smits, Marcel; Hooijdonk, van Toon; Vervoort, Jacques; Hettinga, Kasper

    2016-01-01

    Milk proteins have been shown to be very sensitive to processing. This study aims to investigate the changes of the bovine, camel, and caprine milk proteins after freezing, pasteurization (62 °C, 30 min), and spray drying by proteomic techniques, filter-aided sample preparation (FASP) and

  4. 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% ...

  5. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

    Science.gov (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata

    2018-03-28

    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. 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.

  7. Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Meulendyke, Kelly Ann; Wurth, Mark Allen; McCann, Richard O.; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004)...

  8. Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

    Science.gov (United States)

    Nuez, B; Rojo, F; Salas, M

    1992-12-01

    Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

  9. Processing of Chlamydia abortus polymorphic membrane protein 18D during the chlamydial developmental cycle.

    Science.gov (United States)

    Wheelhouse, Nick M; Sait, Michelle; Aitchison, Kevin; Livingstone, Morag; Wright, Frank; McLean, Kevin; Inglis, Neil F; Smith, David G E; Longbottom, David

    2012-01-01

    Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D). Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae. This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.

  10. Processing of Chlamydia abortus polymorphic membrane protein 18D during the chlamydial developmental cycle.

    Directory of Open Access Journals (Sweden)

    Nick M Wheelhouse

    Full Text Available Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps. While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D.Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae.This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.

  11. 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...... on 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...... provides one of the most comprehensive sets of individual PTM site regulation data for mammalian brain tissue. This will provide a valuable resource for those wishing to perform comparisons or meta-analyses of large scale PTMomic data, as are becoming increasingly common. Furthermore, being focussed...

  12. Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

    Science.gov (United States)

    Banci, Lucia; Bertini, Ivano; Calderone, Vito; Ciofi-Baffoni, Simone; Giachetti, Andrea; Jaiswal, Deepa; Mikolajczyk, Maciej; Piccioli, Mario; Winkelmann, Julia

    2013-01-01

    Biogenesis of iron–sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron–sulfur protein assembly machinery, two human key proteins—NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin—form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron–sulfur cluster proteins. The Ndor1–anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells. PMID:23596212

  13. Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

    DEFF Research Database (Denmark)

    Belsham, Graham

    , open reading frame that encodes a polyprotein. The intact polyprotein is never observed as it is processed, both during and after translation, to 15 different mature proteins plus a variety of precursors. The FMDV capsid protein precursor, P1-2A, is cleaved by the virus encoded 3C protease (3Cpro......) to generate VP0, VP3, VP1 and the peptide 2A. Sixty copies of each of the capsid proteins “self-assemble” into empty capsid particles or with the RNA genome into infectious viruses. These particles normally lack 2A but it is possible to construct and isolate mutant FMDVs in which the cleavage of the VP1/2A...... precursor enhances the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells. Such particles can potentially form the basis of a vaccine but they may only have the same properties as the current inactivated vaccines. We have expressed the FMDV P1-2A alone...

  14. A new theoretical approach to analyze complex processes in cytoskeleton proteins.

    Science.gov (United States)

    Li, Xin; Kolomeisky, Anatoly B

    2014-03-20

    Cytoskeleton proteins are filament structures that support a large number of important biological processes. These dynamic biopolymers exist in nonequilibrium conditions stimulated by hydrolysis chemical reactions in their monomers. Current theoretical methods provide a comprehensive picture of biochemical and biophysical processes in cytoskeleton proteins. However, the description is only qualitative under biologically relevant conditions because utilized theoretical mean-field models neglect correlations. We develop a new theoretical method to describe dynamic processes in cytoskeleton proteins that takes into account spatial correlations in the chemical composition of these biopolymers. Our approach is based on analysis of probabilities of different clusters of subunits. It allows us to obtain exact analytical expressions for a variety of dynamic properties of cytoskeleton filaments. By comparing theoretical predictions with Monte Carlo computer simulations, it is shown that our method provides a fully quantitative description of complex dynamic phenomena in cytoskeleton proteins under all conditions.

  15. 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)

  16. Online integrity monitoring in the protein A step of mAb production processes-increasing reliability and process robustness.

    Science.gov (United States)

    Bork, Christopher; Holdridge, Sarah; Walter, Mark; Fallon, Eric; Pohlscheidt, Michael

    2014-01-01

    The purification of recombinant proteins and antibodies using large packed-bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations-especially of critical process parameters-is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers.

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

    International Nuclear Information System (INIS)

    Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

    2007-01-01

    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

  18. Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases.

    Science.gov (United States)

    Dürrschmid, Karin; Marzban, Gorji; Dürrschmid, Eberhard; Striedner, Gerald; Clementschitsch, Franz; Cserjan-Puschmann, Monika; Bayer, Karl

    2003-01-01

    The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3-10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.

  19. PMA: Protein Microarray Analyser, a user-friendly tool for data processing and normalization.

    Science.gov (United States)

    Da Gama Duarte, Jessica; Goosen, Ryan W; Lawry, Peter J; Blackburn, Jonathan M

    2018-02-27

    Protein microarrays provide a high-throughput platform to measure protein interactions and associated functions, and can aid in the discovery of cancer biomarkers. The resulting protein microarray data can however be subject to systematic bias and noise, thus requiring a robust data processing, normalization and analysis pipeline to ensure high quality and robust results. To date, a comprehensive data processing pipeline is yet to be developed. Furthermore, a lack of analysis consistency is evident amongst different research groups, thereby impeding collaborative data consolidation and comparison. Thus, we sought to develop an accessible data processing tool using methods that are generalizable to the protein microarray field and which can be adapted to individual array layouts with minimal software engineering expertise. We developed an improved version of a previously developed pipeline of protein microarray data processing and implemented it as an open source software tool, with particular focus on widening its use and applicability. The Protein Microarray Analyser software presented here includes the following tools: (1) neighbourhood background correction, (2) net intensity correction, (3) user-defined noise threshold, (4) user-defined CV threshold amongst replicates and (5) assay controls, (6) composite 'pin-to-pin' normalization amongst sub-arrays, and (7) 'array-to-array' normalization amongst whole arrays.

  20. Improved protein hydrogen/deuterium exchange mass spectrometry platform with fully automated data processing.

    Science.gov (United States)

    Zhang, Zhongqi; Zhang, Aming; Xiao, Gang

    2012-06-05

    Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

  1. HPDB-Haskell library for processing atomic biomolecular structures in Protein Data Bank format.

    Science.gov (United States)

    Gajda, Michał Jan

    2013-11-23

    Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The proposed library also features a convenient iterator mechanism, and a simple API modeled after BioPython. I set a new standard for convenience and efficiency of Protein Data Bank processing in a Haskell library, and release it to open source.

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

    OpenAIRE

    Tatiana Coura Oliveira; Samuel Lopes Lima; Josefina Bressan

    2013-01-01

    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 pr...

  3. Aggregation of egg white proteins with pulsed electric fields and thermal processes.

    Science.gov (United States)

    Wu, Li; Zhao, Wei; Yang, Ruijin; Yan, Wenxu; Sun, Qianyan

    2016-08-01

    Pulsed electric field (PEF) processing is progressing towards application for liquid egg to ensure microbial safety. However, it usually causes protein aggregation, and the mechanism is still unclear. In this study, egg white protein was applied to investigate the changes in protein structure and mechanism of aggregates formation and a comparison was made with thermal treatment. Soluble protein content decreased with the increase of turbidity after both treatments. Fluorescence intensity and free sulfhydryl content were increased after being treated at 70 °C for 4 min. Less-remarkable changes of hydrophobicity were observed after PEF treatments (30 kV cm(-1) , 800 µs). Soluble and insoluble aggregates were observed by thermal treatment, and disulfide bonds were the main binding forces. The main components of insoluble aggregates formed by thermal treatment were ovotransferrin (30.58%), lysozyme (18.47%) and ovalbumin (14.20%). While only insoluble aggregates were detected during PEF processes, which consists of ovotransferrin (11.86%), lysozyme (21.11%) and ovalbumin (31.07%). Electrostatic interaction played a very important role in the aggregates formation. PEF had a minor impact on the structure of egg white protein. PEF had insignificant influence on heat-sensitive protein, indicating that PEF has potential in processing food with high biological activity and heat sensitive properties. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  4. Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion

    Directory of Open Access Journals (Sweden)

    Magdalena Montowska

    2016-01-01

    Full Text Available New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages. Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed aft er 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

  5. Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Mader Diana

    2013-01-01

    Full Text Available Abstract Background Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. Results In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. Conclusions These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

  6. Towards protein crystallization as a process step in downstream processing of therapeutic antibodies: screening and optimization at microbatch scale.

    Directory of Open Access Journals (Sweden)

    Yuguo Zang

    Full Text Available Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4 antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step.

  7. Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.

    Science.gov (United States)

    Buyel, Johannes Felix; Fischer, Rainer

    2014-01-01

    All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilitating the separation of solids and liquids, and thus reducing process costs. However, many different factors can affect the behavior of flocculants, including the pH and conductivity of the medium, the size and charge distribution of the particulates, and the charge density and molecular mass of the polymer. Importantly, these properties can also affect the recovery of the target protein and the overall safety profile of the process. We therefore used a design of experiments approach to establish reliable predictive models that characterize the impact of flocculants during the downstream processing of biopharmaceutical proteins. We highlight strategies for the selection of flocculants during process optimization. These strategies will contribute to the quality by design aspects of process development and facilitate the development of safe and efficient downstream processes for plant-derived pharmaceutical proteins.

  8. The Protein Disulfide Isomerase of Botrytis cinerea: An ER Protein Involved in Protein Folding and Redox Homeostasis Influences NADPH Oxidase Signaling Processes

    Directory of Open Access Journals (Sweden)

    Robert Marschall

    2017-05-01

    Full Text Available Botrytis cinerea is a filamentous plant pathogen, which infects hundreds of plant species; within its lifestyle, the production of reactive oxygen species (ROS and a balanced redox homeostasis are essential parameters. The pathogen is capable of coping with the plant’s oxidative burst and even produces its own ROS to enhance the plant’s oxidative burst. Highly conserved NADPH oxidase (Nox complexes produce the reactive molecules. The membrane-associated complexes regulate a large variety of vegetative and pathogenic processes. Besides their commonly accepted function at the plasma membrane, recent studies reveal that Nox complexes are also active at the membrane of the endoplasmic reticulum. In this study, we identified the essential ER protein BcPdi1 as new interaction partner of the NoxA complex in B. cinerea. Mutants that lack this ER chaperone display overlapping phenotypes to mutants of the NoxA signaling pathway. The protein appears to be involved in all major developmental processes, such as the formation of sclerotia, conidial anastomosis tubes and infection cushions (IC’s and is needed for full virulence. Moreover, expression analyses and reporter gene studies indicate that BcPdi1 affects the redox homeostasis and unfolded protein response (UPR-related genes. Besides the close association between BcPdi1 and BcNoxA, interaction studies provide evidence that the ER protein might likewise be involved in Ca2+ regulated processes. Finally, we were able to show that the potential key functions of the protein BcPdi1 might be affected by its phosphorylation state.

  9. ChIP-seq Data Processing for PcG Proteins and Associated Histone Modifications.

    Science.gov (United States)

    Bogdanovic, Ozren; van Heeringen, Simon J

    2016-01-01

    Chromatin Immunoprecipitation followed by massively parallel DNA sequencing (ChIP-sequencing) has emerged as an essential technique to study the genome-wide location of DNA- or chromatin-associated proteins, such as the Polycomb group (PcG) proteins. After being generated by the sequencer, raw ChIP-seq sequence reads need to be processed by a data analysis pipeline. Here we describe the computational steps required to process PcG ChIP-seq data, including alignment, peak calling, and downstream analysis.

  10. 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

    : 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...... was followed by determination of protein solubility, protein thiol groups and protein carbonyl groups using colorimetric methods as well as western blotting for protein carbonyl groups. Lipid and protein oxidation markers indicated that both lipid and protein oxidation took place during processing...

  11. 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.

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

    International Nuclear Information System (INIS)

    El-Niely, Hania F.G.

    2007-01-01

    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

  13. Single cell protein production of Chlorella sp. using food processing waste as a cultivation medium

    Science.gov (United States)

    Putri, D.; Ulhidayati, A.; Musthofa, I. A.; Wardani, A. K.

    2018-03-01

    The aim of this study was to investigate the effect of various food processing wastes on the production of single cell protein by Chlorella sp. Three various food processing wastes i.e. tofu waste, tempeh waste and cheese whey waste were used as cultivation medium for Chlorella sp. growth. Sea water was used as a control of cultivation medium. The addition of waste into cultivation medium was 10%, 20%, 30%, 40%, and 50%. The result showed that the highest yield of cell mass and protein content was found in 50% tofu waste cultivation medium was 47.8 × 106 cell/ml with protein content was 52.24%. The 50% tofu waste medium showed improved cell yield as nearly as 30% than tempeh waste medium. The yield of biomass and protein content when 30% tempeh waste was used as cultivation medium was 37.1 × 106 cell/ml and 52%, respectively. Thus, food processing waste especially tofu waste would be a promising candidate for cultivation medium for single cell production from Chlorella sp. Moreover, the utilization of waste can reduce environmental pollution and increase protein supply for food supplement or animal feed.

  14. A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

    Science.gov (United States)

    Shibahara, Yusuke; Uesaka, Yoshihiko; Wang, Jun; Yamada, Shoichi; Shiomi, Kazuo

    2013-01-15

    Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 μg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. RECOVERY OF PROTEIN FROM MUNG BEAN STARCH PROCESSING WASTEWATER BY ROTATING ULTRAFILTRATION

    Directory of Open Access Journals (Sweden)

    PENPORN SRINIWORN

    2016-07-01

    Full Text Available Mung bean wastewater containing valuable protein is very potential to be recovered for reuse. In this study, rotary disk ultrafiltration was employed to recover this protein. The effects of transmembrane pressure (TMP and membrane rotational speeds on process efficiency were studied and the optimum condition was chosen based on membrane permeate flux and protein retention. The results suggested that the use of TMP of 1.2 bar and rotating speed of 1,683 rpm under total recycle mode tended to achieve highest permeate flux (43 L/m3h compared to those using lower TMP and rotating speeds. The permeate fluxes under total recycle mode and batch concentration mode tended to increase with processing time, indicating the effectiveness of rotating shear force. In addition, the effect of stabilization technique on process performance under batch concentration mode was also studied. However, the variable did not show positive impacts on permeate flux and protein retention improvement. The optimum condition to achieve volume concentration factor (VCF of 5 was TMP of 1.2 bar and rotating speed of 1,403 rpm without stabilization. Under this condition, the average flux, protein retention and energy consumption were 42 L/m2h, 96% and 81 kWh/m3, respectively.

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

    International Nuclear Information System (INIS)

    2003-01-01

    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)

  17. 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.

  18. 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

  19. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    Science.gov (United States)

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Effect of Processing Intensity on Immunologically Active Bovine Milk Serum Proteins

    Science.gov (United States)

    Brick, Tabea; Ege, Markus; Böck, Andreas; von Mutius, Erika; Vervoort, Jacques

    2017-01-01

    Consumption of raw cow’s milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general is missing. The aim of this study was to compare the native milk serum proteome between raw cow’s milk and various industrially applied processing methods, i.e., homogenization, fat separation, pasteurization, ultra-heat treatment (UHT), treatment for extended shelf-life (ESL), and conventional boiling. Each processing method was applied to the same three pools of raw milk. Levels of detectable proteins were quantified by liquid chromatography/tandem mass spectrometry following filter aided sample preparation. In total, 364 milk serum proteins were identified. The 140 proteins detectable in 66% of all samples were entered in a hierarchical cluster analysis. The resulting proteomics pattern separated mainly as high (boiling, UHT, ESL) versus no/low heat treatment (raw, skimmed, pasteurized). Comparing these two groups revealed 23 individual proteins significantly reduced by heating, e.g., lactoferrin (log2-fold change = −0.37, p = 0.004), lactoperoxidase (log2-fold change = −0.33, p = 0.001), and lactadherin (log2-fold change = −0.22, p = 0.020). The abundance of these heat sensitive proteins found in higher quantity in native cow’s milk compared to heat treated milk, renders them potential candidates for protection from asthma, allergies, and respiratory infections. PMID:28858242

  1. 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. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  2. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions.

    Science.gov (United States)

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; van Boekel, Martinus; Fogliano, Vincenzo; Stieger, Markus

    2016-09-28

    The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were treated by HPHT processing or conventional high-temperature (HT) treatments. Browning was reduced, and early and advanced Maillard reactions were retarded under HPHT processing at all pH values compared to HT treatment. HPHT induced a larger pH drop than HT treatments, especially at pH 9, which was not associated with Maillard reactions. After HPHT processing at pH 7, protein aggregation and viscosity of whey protein isolate-glucose/trehalose solutions remained unchanged. It was concluded that HPHT processing can potentially improve the quality of protein-sugar-containing foods, for which browning and high viscosities are undesired, such as high-protein beverages.

  3. Advances in structure formation of anisotropic protein-rich foods through novel processing concepts

    NARCIS (Netherlands)

    Manski, J.M.; Goot, van der A.J.; Boom, R.M.

    2007-01-01

    Development of protein-rich food products is currently limited by lack of scientific insights in structuring processes. The application of well-defined flow appears to be a good tool to create novel anisotropic food structures, on one hand, and to improve understanding of the behavior of

  4. Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration

    NARCIS (Netherlands)

    Safi, C.; Olivieri, G.; Pina Campos, Rui; Engelen-Smit, N.; Mulder, W.J.; Broek, van den L.A.M.; Sijtsma, L.

    2017-01-01

    A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100 g.L−1 solution of cells was first subjected to cell disruption by either high-pressure

  5. 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. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrome

  6. Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography.

    Science.gov (United States)

    Lin, Miao-Fang; Royal, Mabel; Hayenga, Kirk; Conn, Greg

    2003-07-25

    We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.

  7. Enhanced functionality of pea-rice protein isolate blends through direct steam injection processing.

    Science.gov (United States)

    Pietrysiak, Ewa; Smith, Denise M; Smith, Brennan M; Ganjyal, Girish M

    2018-03-15

    Direct steam injection (DSI) processing with pH adjustment was investigated to enhance the functionality of pea-rice protein isolate blends (PR). Protein slurries at concentration of 5%(w/w) of commercial pea and rice protein isolates in the ratio of 2:1(w/w) across a range of steam temperatures (66-107°C) and pH values (2-11) were studied. After DSI treatment, the PR were freeze-dried to obtain the final dry protein powder. Based on protein solubility profiles, the optimal DSI processing conditions were 107°C and pH 11. Available lysine was not reduced (P>0.05) in the blend. Solubility (from 3 to 41%, at pH 7), emulsifying activity index (from 5.9 to 52.5m 2 /g), foam stability (from 68.2 to 82.8%), and oil holding capacity (from 1.8 to 4.9g/g) values increased (P<0.05) compared to the untreated PR. DSI can modify the functionality of PR without affecting the essential amino acid composition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Microbial Protein Production from Candida tropicalis ATCC13803 in a Submerged Batch Fermentation Process

    Directory of Open Access Journals (Sweden)

    Sahar Golaghaiee

    2017-01-01

    Full Text Available Background and Objective: Microbial protein production can resolve one of the major world challenges, i.e. lack of protein sources. Candida tropicalis growth was investigated to specify a medium to reach the highest cell proliferation and protein production.Material and Methods: Fractional factorial design and the index of signal to noise ratio were applied for optimization of microbial protein production. Optimization process was conducted based on the experimental results of Taguchi approach designs. Fermentationwas performed at 25oC and the agitation speed of 300 rpm for 70 h. Ammonium sulfate, iron sulfate, glycine and glucose concentrations were considered as process variables. Optimization of the culture medium composition was conducted in order to obtain the highest cell biomass concentration and protein content. Experiment design was performed based on the Taguchi approach and L-16 orthogonal arrays using Qualitek-4 software.Results and Conclusion: Maximum biomass of 8.72 log (CFU ml-1 was obtained using the optimized medium with 0.3, 0.15, 2 and 80 g l-1 of ammonium sulfate, iron sulfate, glycine and glucose, respectively. Iron sulfate and ammonium sulfate with 41.76% (w w-1 and 35.27% (w w-1 contributions, respectively, were recognized as the main components for cell growth. Glucose and glycine with 17.12% and 5.86% (w w-1 contributions,respectively, also affected cell production. The highest interaction severity index of +54.16% was observed between glycine and glucose while the least one of +0.43% was recorded for ammonium sulfate and glycine. A deviation of 7% between the highestpredicted cell numbers and the experimented count confirms the suitability of the applied statistical method. High protein content of 52.16% (w w-1 as well as low fat and nucleic acids content suggest that Candida tropicalis is a suitable case for commercial processes.Conflict of interest: The authors declare that there is no conflict of interest.

  9. Enhancement of Protein and Pigment Content in Two Chlorella Species Cultivated on Industrial Process Water

    Directory of Open Access Journals (Sweden)

    Hamed Safafar

    2016-12-01

    Full Text Available Chlorella pyrenoidosa and Chlorella vulgaris were cultivated in pre-gasified industrial process water with high concentration of ammonia representing effluent from a local biogas plant. The study aimed to investigate the effects of growth media and cultivation duration on the nutritional composition of biomass. Variations in proteins, lipid, fatty acid composition, amino acids, tocopherols, and pigments were studied. Both species grew well in industrial process water. The contents of proteins were affected significantly by the growth media and cultivation duration. Microalga Chlorella pyrenoidosa produced the highest concentrations of protein (65.2% ± 1.30% DW while Chlorella vulgaris accumulated extremely high concentrations of lutein and chlorophylls (7.14 ± 0.66 mg/g DW and 32.4 ± 1.77 mg/g DW, respectively. Cultivation of Chlorella species in industrial process water is an environmentally friendly, sustainable bioremediation method with added value biomass production and resource valorization, since the resulting biomass also presented a good source of proteins, amino acids, and carotenoids for potential use in aquaculture feed industry.

  10. Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice

    Science.gov (United States)

    Sahly, Iman; Dufour, Eric; Schietroma, Cataldo; Michel, Vincent; Bahloul, Amel; Perfettini, Isabelle; Pepermans, Elise; Estivalet, Amrit; Carette, Diane; Aghaie, Asadollah; Ebermann, Inga; Lelli, Andrea; Iribarne, Maria; Hardelin, Jean-Pierre; Weil, Dominique; Sahel, José-Alain

    2012-01-01

    The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins—myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans—do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner–outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients. PMID:23045546

  11. Processing of recombinant Listeria monocytogenes proteins for MHC class I presentation follows a dedicated, high-efficiency pathway

    Science.gov (United States)

    Wolf, Benjamin J.; Princiotta, Michael F.

    2013-01-01

    CD8+ T lymphocytes recognize short peptides of ~8–10 amino acids bound to MHC class I molecules (pMHC) on the surface of antigen presenting cells. These peptides can be generated from either endogenous proteins synthesized by the biosynthetic machinery of the presenting cell or from exogenously sourced proteins. Because much of the research characterizing the MHC class I processing pathway has focused on endogenously synthesized proteins, it is not known whether differences exist in the processing pathway followed by endogenously synthesized versus exogenously sourced proteins. To highlight potential differences in the processing of endogenous versus exogenous proteins, we developed a model system to measure the efficiency of pMHC generation from nearly identical recombinant proteins expressed from vaccinia virus and Listeria monocytogenes. In these experiments, we uncovered a striking difference in the way recombinant Listeria antigens are processed and presented when compared to endogenously synthesized viral proteins. Specifically, we find that pMHC production from secreted Listeria proteins occurs at the same rate, independent of the cellular half-life of the protein from which it is derived, whereas the rate of pMHC production from endogenously synthesized viral proteins is absolutely dependent on its protein half-life. Accordingly, our data demonstrate the existence of a distinct and highly efficient MHC class I presentation pathway used for the processing of at least some exogenously synthesized proteins. PMID:23396941

  12. Discrepancy between mRNA and protein abundance: insight from information retrieval process in computers.

    Science.gov (United States)

    Wang, Degeng

    2008-12-01

    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 information flow from storage level to the execution level. Functional similarities can be found in almost every facet of the retrieval process. Firstly, common architecture is shared, as the ribonome (RNA space) and the proteome (protein space) are functionally similar to the computer primary memory and the computer cache memory, respectively. Secondly, the retrieval process functions, in both systems, to support the operation of dynamic networks-biochemical regulatory networks in cells and, in computers, the virtual networks (of CPU instructions) that the CPU travels through while executing computer programs. Moreover, many regulatory techniques are implemented in computers at each step of the information retrieval process, with a goal of optimizing system performance. Cellular counterparts can be easily identified for these regulatory techniques. In other words, this comparative study attempted to utilize theoretical insight from computer system design principles as catalysis to sketch an integrative view of the gene expression process, that is, how it functions to ensure efficient operation of the overall cellular regulatory network. In context of this bird's-eye view, discrepancy between protein and RNA abundance became a logical observation one would expect. It was suggested that this discrepancy, when interpreted in the context of system operation, serves as a potential source of information to decipher regulatory logics underneath biochemical network operation.

  13. UV Irradiation Accelerates Amyloid Precursor Protein (APP) Processing and Disrupts APP Axonal Transport

    Science.gov (United States)

    Almenar-Queralt, Angels; Falzone, Tomas L.; Shen, Zhouxin; Lillo, Concepcion; Killian, Rhiannon L.; Arreola, Angela S.; Niederst, Emily D.; Ng, Kheng S.; Kim, Sonia N.; Briggs, Steven P.; Williams, David S.

    2014-01-01

    Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction. PMID:24573290

  14. Effect of processing on protein digestibility, biological value and net protein utilization of millet and legume based infant mixes and biscuits.

    Science.gov (United States)

    Geervani, P; Vimala, V; Pradeep, K U; Devi, M R

    1996-04-01

    Effect of combinations of millet and legume and processing on digestibility, biological value and net protein utilization was evaluated using albino rats. The millets and legumes selected for the study include sorghum, pearl millet, finger millet, chickpea and green gram (P radiatus). The processes tested include dehulling, boiling, roasting, malting and baking. Among the combinations tested, the sorghum-chickpea combination had significantly (p < 0.05) higher digestibility. Between the processes tested, roasting resulted in significantly higher net protein utilization. Results of biological study on biscuits prepared by using millet and legume combination flours, indicated the biscuits to be of good protein quality.

  15. From Raw Data to Protein Backbone Chemical Shifts Using NMRFx Processing and NMRViewJ Analysis.

    Science.gov (United States)

    Johnson, Bruce A

    2018-01-01

    Assignment of the chemical shifts of the backbone atoms (HN, N, CA, CB, and C) of proteins is often a prerequisite to using NMR information in the study of proteins. These chemical shifts and their perturbations are the basis for the analysis of protein dynamics, ligand binding, and backbone conformation. They are generally assigned prior to full side-chain assignments and the determination of the complete three-dimensional molecular structure. This chapter describes the use of two software packages, NMRFx Processor and NMRViewJ, in going from raw NMR data to backbone assignments. The step-by-step procedure describes processing of the data and the use of manual and automated features of the RunAbout tool in NMRViewJ to perform the assignments.

  16. 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.

  17. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine; Xi, Bingyan; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Fogliano, Vincenzo; Stieger, Markus

    2016-01-01

    The aim of the study was to determine the influence of pressure in high-pressure-high-temperature (HPHT) processing on Maillard reactions and protein aggregation of whey protein-sugar solutions. Solutions of whey protein isolate containing either glucose or trehalose at pH 6, 7, and 9 were

  18. Markovian and non-Markovian protein sequence evolution: aggregated Markov process models.

    Science.gov (United States)

    Kosiol, Carolin; Goldman, Nick

    2011-08-26

    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 timescales. However, any long time period is simply the accumulation of many short time periods, and it remains unclear why evolution should appear to act systematically differently across the range of timescales studied. We show that the observed time-dependent behavior can be explained qualitatively by modeling protein sequence evolution as an aggregated Markov process (AMP): a time-homogeneous Markovian substitution model observed only at the level of the amino acids encoded by the protein-coding DNA sequence. The study of AMPs sheds new light on the relationship between amino acid-level and codon-level models of sequence evolution, and our results suggest that protein evolution should be modeled at the codon level rather than using amino acid substitution models. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Protein carbonylation sites in bovine raw milk and processed milk products.

    Science.gov (United States)

    Milkovska-Stamenova, Sanja; Mnatsakanyan, Ruzanna; Hoffmann, Ralf

    2017-08-15

    During thermal treatment of milk, proteins are oxidized, which may reduce the nutritional value of milk, abolish protein functions supporting human health, especially important for newborns, and yield potentially harmful products. The side chains of several amino acids can be oxidized to reactive carbonyls, which are often used to monitor oxidative stress in organisms. Here we mapped protein carbonylation sites in raw milk and different brands of pasteurized, ultra high temperature (UHT) treated milk, and infant formulas (IFs) after digesting the precipitated proteins with trypsin. Reactive carbonyls were derivatized with O-(biotinylcarbazoylmethyl)hydroxylamine to enrich the modified peptides by avidin-biotin affinity chromatography and analyze them by nanoRP-UPLC-ESI-MS. Overall, 53 unique carbonylated peptides (37 carbonylation sites, 15 proteins) were identified. Most carbonyls were derived from dicarbonyls (mainly glyoxal). The number of carbonylation sites increased with the harsher processing from raw milk (4) to pasteurized (16) and UHT milk (16) and to IF (24). Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

    Science.gov (United States)

    Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

    2014-08-01

    Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. 14-3-3 Proteins regulate exonuclease 1-dependent processing of stalled replication forks.

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    Kim Engels

    2011-04-01

    Full Text Available Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1 processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress.

  3. Improved process analytical technology for protein a chromatography using predictive principal component analysis tools.

    Science.gov (United States)

    Hou, Ying; Jiang, Canping; Shukla, Abhinav A; Cramer, Steven M

    2011-01-01

    Protein A chromatography is widely employed for the capture and purification of antibodies and Fc-fusion proteins. Due to the high cost of protein A resins, there is a significant economic driving force for using these chromatographic materials for a large number of cycles. The maintenance of column performance over the resin lifetime is also a significant concern in large-scale manufacturing. In this work, several statistical methods are employed to develop a novel principal component analysis (PCA)-based tool for predicting protein A chromatographic column performance over time. A method is developed to carry out detection of column integrity failures before their occurrence without the need for a separate integrity test. In addition, analysis of various transitions in the chromatograms was also employed to develop PCA-based models to predict both subtle and general trends in real-time protein A column yield decay. The developed approach has significant potential for facilitating timely and improved decisions in large-scale chromatographic operations in line with the process analytical technology (PAT) guidance from the Food and Drug Administration (FDA). © 2010 Wiley Periodicals, Inc.

  4. Economic comparison of multiple techniques for recovering leaf protein in biomass processing.

    Science.gov (United States)

    Bals, Bryan; Dale, Bruce E

    2011-03-01

    Leaf protein concentrates (LPC) can be used as a valuable co-product to cellulosic biofuel production and can also mitigate the food versus fuel controversy. Two major approaches have been considered for LPC production: a well-characterized mechanical pressing method and a less studied method involving aqueous extraction with recovery using ultrafiltration. Experimental results with switchgrass extracts show low protein recovery after filtration, particularly if protein is recovered after cellulose hydrolysis. Economic modeling suggests that aqueous extraction costs less than mechanical pressing, but due to lower protein yields and lower quality, overall profit is higher for mechanical pressing versus aqueous extraction ($26/Mg feedstock vs. $14/Mg). If modest improvements can be made in extraction yields, filtration recovery, and protein quality, then the profitability of the aqueous extraction approach can be increased to $37/Mg feedstock. This study suggests that aqueous extraction is a viable alternative for LPC co-production in a biorefinery if key improvements can be made in the process. Copyright © 2010 Wiley Periodicals, Inc.

  5. Amyloid Precursor Proteins Are Dynamically Trafficked and Processed During Neuronal Development

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    Jenna M. Ramaker

    2016-11-01

    Full Text Available Proteolytic processing of the Amyloid Precursor Protein (APP produces beta-amyloid (Aβ peptide fragments that accumulate in Alzheimer’s Disease (AD, but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2 that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta, we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate

  6. A unique mechanism for protein processing and degradation in Arabidopsis thaliana

    Science.gov (United States)

    Rojo, Enrique; Zouhar, Jan; Carter, Clay; Kovaleva, Valentina; Raikhel, Natasha V.

    2003-01-01

    Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report in vivo evidence that the precursor protease vesicle-localized vacuolar processing enzyme-γ (VPEγ) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPEγ is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpeγ mutants but not in WT plants, suggesting a unique role of VPEγ in protein processing and degradation in Arabidopsis. PMID:12773619

  7. Food Processing: The Influence of the Maillard Reaction on Immunogenicity and Allergenicity of Food Proteins

    Directory of Open Access Journals (Sweden)

    Malgorzata Teodorowicz

    2017-08-01

    Full Text Available The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR. Maillard reaction products (MRPs contribute to the taste, smell and color of many food products, and thus influence consumers’ choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs. This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins.

  8. Food Processing: The Influence of the Maillard Reaction on Immunogenicity and Allergenicity of Food Proteins.

    Science.gov (United States)

    Teodorowicz, Malgorzata; van Neerven, Joost; Savelkoul, Huub

    2017-08-04

    The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR). Maillard reaction products (MRPs) contribute to the taste, smell and color of many food products, and thus influence consumers' choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs). This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins.

  9. Thermal unfolding of eosinophil cationic protein/ribonuclease 3: A nonreversible process

    OpenAIRE

    Nikolovski, Zoran; Buzón, Víctor; Ribó, Marc; Moussaoui, Mohammed; Vilanova, Maria; Cuchillo, Claudi M.; Cladera, Josep; Nogués, M. Victòria

    2006-01-01

    Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth-derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T 1/2 val...

  10. DEVELOPMENT OF TECHNOLOGY FOR WHEAT PROCESSING INTO ALCOHOL AND PROTEIN PRODUCT

    Directory of Open Access Journals (Sweden)

    T. I. Romanyuk

    2015-01-01

    Full Text Available In the alcohol industry it is important to create non-waste technology for grain processing into alcohol. The aim of research was the development of technology for wheat processing into ethanol and protein product. We studied the process of enzymatic hydrolysis of starch with glucoamylase of Glucogam preparation. We determined the optimal dosage of the enzyme 8 units. GlA/g of starch, and the temperature of 55°C. In the study of protein hydrolysis by the concomitant to glucoamylase protease of enzyme Glucogam preparation accumulation of amino nitrogen of 4.5 mg / cm 3 in 7 hours of bioconversion takes place. Separation of the resulting saccharified mass was carried out by centrifugation into the filtrate and protein mass. Centrifugation was carried out at a rotational speed of 2500 rev / min for 8 minutes. Protein was dried to 5% moisture content at temperatures not exceeding 35°C, milled, and examined its properties in comparison with native wheat gluten. The resulting product had the following characteristics: the solubility of 10%, water-holding capacity of 1.53 g / g, and fat binding capacity of 1.9 g /g. We investigated the process of fermentation of clarified wort with the dry solids concentration of 14%. We used the yeast Saccharomyces cerevisiae of race XII and Saccharomyces cerevisiae of race IMB Y-5007 in the dose of 120 million cells per 1 cm3 of wort. Optimum composition of mineral salts was determined. For the yeasts of race XII and IMB Y-5007 fertilizing with diammonium phosphate in a dosage of 1.5 g / dm3 is necessary. The alcohol yield when using the yeasts of race IMB Y-5007 was 60.7 dal/ ton of conditional starch, when using yeasts of race XII it accounts 60,6 dal / ton of conditional starch.

  11. Protein Viability on Au Nanoparticles during an Electrospray and Electrostatic-Force-Directed Assembly Process

    Directory of Open Access Journals (Sweden)

    Shun Mao

    2010-01-01

    Full Text Available We study the protein viability on Au nanoparticles during an electrospray and electrostatic-force-directed assembly process, through which Au nanoparticle-antibody conjugates are assembled onto the surface of carbon nanotubes (CNTs to fabricate carbon nanotube field-effect transistor (CNTFET biosensors. Enzyme-linked immunosorbent assay (ELISA and field-effect transistor (FET measurements have been used to investigate the antibody activity after the nanoparticle assembly. Upon the introduction of matching antigens, the colored reaction from the ELISA and the change in the electrical characteristic of the CNTFET device confirm that the antibody activity is preserved during the assembly process.

  12. Simple control of fed-batch processes for recombinant protein production with E. coli.

    Science.gov (United States)

    Schaepe, Sebastian; Kuprijanov, Artur; Aehle, Mathias; Simutis, Rimvydas; Lübbert, Andreas

    2011-09-01

    A very simple but effective process control technique is proposed that leads to a high batch-to-batch reproducibility with respect to biomass concentration as well as the specific biomass growth rate profiles in E. coli fermentations performed during recombinant protein production. It makes use of the well-established temperature controllers in currently used fermenters, but takes its information from the difference between the controlled culture temperature T (cult) and the temperature T (coolin) of the coolant fed to the fermenter's cooling jacket as adjusted by the fermenter temperature controller. For process control purposes this measured difference is corrected regarding stirrer influences and cumulated before it is used as a new process control variable. As a spin-off of this control, it becomes possible to estimate online the oxygen mass transfer rates and the corresponding k(L)a values during the real cultivation process. © Springer Science+Business Media B.V. 2011

  13. Prion removal capacity of plasma protein manufacturing processes: a data collection from PPTA member companies.

    Science.gov (United States)

    Cai, Kang; Gröner, Albrecht; Dichtelmüller, Herbert O; Fabbrizzi, Fabrizio; Flechsig, Eckhard; Gajardo, Rodrigo; von Hoegen, Ilka; Jorquera, Juan I; Kempf, Christoph; Kreil, Thomas R; Lee, Douglas C; Moscardini, Mila; Pölsler, Gerhard; Roth, Nathan J

    2013-09-01

    The variant Creutzfeldt-Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large-scale manufacturing of human plasma-derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety. Collectively analyzing the results could reveal experimental reproducibility and detect trends and mechanisms driving prion removal. Plasma Protein Therapeutics Association member companies collected more than 200 prion removal studies on plasma protein manufacturing steps, including precipitation, adsorption, chromatography, and filtration, as well as combined steps. The studies used a range of model spiking agents and bench-scale process replicas. The results were grouped based on key manufacturing variables to identify factors impacting removal. The log reduction values of a group are presented for comparison. Overall prion removal capacities evaluated by independent groups were in good agreement. The removal capacity evaluated using biochemical assays was consistent with prion infectivity removal measured by animal bioassays. Similar reduction values were observed for a given step using various spiking agents, except highly purified prion protein in some circumstances. Comparison between combined and single-step studies revealed complementary or overlapping removal mechanisms. Steps with high removal capacities represent the conditions where the physiochemical differences between prions and therapeutic proteins are most significant. The results support the intrinsic ability of certain plasma protein manufacturing steps to remove prions in case of an unlikely contamination, providing a safeguard to products. © 2012 American Association of Blood Banks.

  14. 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. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. PENGARUH PENAMBAHAN MAGNESIUM STEARAT DAN JENIS PROTEIN PADA PEMBUATAN BIODEGRADABLE FOAM DENGAN METODE BAKING PROCESS

    Directory of Open Access Journals (Sweden)

    Nanik hendrawati

    2015-12-01

    Full Text Available Biodegradable foam with cassava starch, protein and chitosan as the basic ingredients can be produced by using baking process method. Variation on magnesium stearate amount and protein types gave different effect on the biodegradable foam quality. The amount of magnesium stearate was varied as 1; 1.6; 2.2; 2.8; 3.4 and 4 % w/w and the sources of protein used in this research were taken from soy bean, peanut and egg white. The foam produced in this research was then tested for its mechanical properties, water resistance and biodegradability. It was found that addition of magnesium stearate as much as 4% w/w reduced water adsorption and biodegradability of foam. Magnesium stearate affected the ability of absorption of water and foam degradation, but did not influence on tensile strength. Different types of protein also gave influence on water absorption, biodegradability and tensile strength. The best improvement of tensile strenght among the compounds tested was shown by soy bean based foam.

  16. 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.

  17. 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.

  18. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.

    Science.gov (United States)

    Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A; Salic, Adrian

    2011-03-07

    The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly.

  19. 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.

  20. FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation.

    Science.gov (United States)

    Popow, Johannes; Alleaume, Anne-Marie; Curk, Tomaz; Schwarzl, Thomas; Sauer, Sven; Hentze, Matthias W

    2015-11-01

    Mitochondrial RNA processing is an essential step for the synthesis of the components of the electron transport chain in all eukaryotic organisms, yet several aspects of mitochondrial RNA biogenesis and regulation are not sufficiently understood. RNA interactome capture identified several disease-relevant RNA-binding proteins (RBPs) with noncanonical RNA-binding architectures, including all six members of the FASTK (FAS-activated serine/threonine kinase) family of proteins. A mutation within one of these newly assigned FASTK RBPs, FASTKD2, causes a rare form of Mendelian mitochondrial encephalomyopathy. To investigate whether RNA binding of FASTKD2 contributes to the disease phenotype, we identified the RNA targets of FASTKD2 by iCLIP. FASTKD2 interacts with a defined set of mitochondrial transcripts including 16S ribosomal RNA (RNR2) and NADH dehydrogenase subunit 6 (ND6) messenger RNA. CRISPR-mediated deletion of FASTKD2 leads to aberrant processing and expression of RNR2 and ND6 mRNA that encodes a subunit of the respiratory complex I. Metabolic phenotyping of FASTKD2-deficient cells reveals impaired cellular respiration with reduced activities of all respiratory complexes. This work identifies key aspects of the molecular network of a previously uncharacterized, disease-relevant RNA-binding protein, FASTKD2, by a combination of genomic, molecular, and metabolic analyses. © 2015 Popow et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration.

    Science.gov (United States)

    Safi, C; Olivieri, G; Campos, R P; Engelen-Smit, N; Mulder, W J; van den Broek, L A M; Sijtsma, L

    2017-02-01

    A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100g.L -1 solution of cells was first subjected to cell disruption by either high-pressure homogenization (HPH) or enzymatic treatment (ENZ). HPH resulted in a larger release of proteins (49%) in the aqueous phase compared to the Alcalase incubation (35%). In both cases, an ultrafiltration/diafiltration (UF/DF) was then performed on the supernatant obtained from cell disruption by testing different membrane cut-off (1000kDa, 500kDa and 300kDa). After optimising the process conditions, the combination of ENZ→UF/DF ended in a larger overall yield of water soluble proteins (24.8%) in the permeate compared to the combination of HPH→UF/DF (17.4%). A gel polarization model was implemented to assess the maximum achievable concentration factor during ultrafiltration and the mass transfer coefficient related to the theoretical permeation flux rate. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Effects of processing on immunoreactivity of cashew nut (Anacardium occidentale L.) seed flour proteins.

    Science.gov (United States)

    Venkatachalam, Mahesh; Monaghan, Erin K; Kshirsagar, Harshal H; Robotham, Jason M; O'Donnell, Susan E; Gerber, Mary Susan; Roux, Kenneth H; Sathe, Shridhar K

    2008-10-08

    Cashew nut seeds were subjected to processing including autoclaving (121 degrees C for 5, 10, 20, and 30 min), blanching (100 degrees C for 1, 4, 7, and 10 min), microwave heating (1 and 2 min each at 500 and 1000 W), dry roasting (140 degrees C for 20 and 30 min; 170 degrees C for 15 and 20 min; and 200 degrees C for 10 and 15 min), gamma-irradiation (1, 5, 10, and 25 kGy), and pH (1, 3, 5, 7, 9, 11, and 13). Proteins from unprocessed and processed cashew nut seeds were probed for stability using anti-Ana o 2 rabbit polyclonal antibodies and mouse monoclonal antibodies directed against Ana o 1, Ana o 2, and Ana o 3 as detection agents. Results indicate that Ana o 1, Ana o 2, and Ana o 3 are stable regardless of the processing method to which the nut seeds are subjected.

  3. Near-infrared reflectance spectroscopy for predicting amino acids content in intact processed animal proteins.

    Science.gov (United States)

    De la Haba, Maria José; Garrido-Varo, Ana; Guerrero-Ginel, José Emilio; Pérez-Marín, Dolores C

    2006-10-04

    Near-infrared calibrations were developed for the instantaneous prediction of amino acids composition of processed animal proteins (PAPs). Two sample presentation modes were compared (ground vs intact) for demonstrating the viability of the analysis in the intact form, avoiding the need for milling. Modified partial least-squares (MPLS) equations for the prediction of amino acids in PAPs were developed using the same set of samples (N = 92 PAPs) analyzed in ground and intact form and in three cups differing in the optical window size. The standard error for cross validation (SECV) and the coefficient of determination (1-VR) values yielded with the calibrations developed using the samples analyzed in the intact form showed similar or even better accuracy than those obtained with finely ground samples. The excellent predictive ability (1-VR > 0.90; CV marketing of these important protein feed ingredients, alleviating the costs and time associated with the routine quality controls.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fuente, Alberto de la; Fotia, Giorgio; Maggio, Fabio; Mancosu, Gianmaria; Pieroni, Enrico [CRS4 Bioinformatica, Parco Tecnologico POLARIS, Ed.1, Loc Piscinamanna, Pula (Italy)], E-mail: alf@crs4.it

    2008-06-06

    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.

  5. Regulation of amyloid precursor protein processing by the Beclin 1 complex.

    Directory of Open Access Journals (Sweden)

    Philipp A Jaeger

    2010-06-01

    Full Text Available Autophagy is an intracellular degradation pathway that functions in protein and organelle turnover in response to starvation and cellular stress. Autophagy is initiated by the formation of a complex containing Beclin 1 (BECN1 and its binding partner Phosphoinositide-3-kinase, class 3 (PIK3C3. Recently, BECN1 deficiency was shown to enhance the pathology of a mouse model of Alzheimer Disease (AD. However, the mechanism by which BECN1 or autophagy mediate these effects are unknown. Here, we report that the levels of Amyloid precursor protein (APP and its metabolites can be reduced through autophagy activation, indicating that they are a substrate for autophagy. Furthermore, we find that knockdown of Becn1 in cell culture increases the levels of APP and its metabolites. Accumulation of APP and APP C-terminal fragments (APP-CTF are accompanied by impaired autophagosomal clearance. Pharmacological inhibition of autophagosomal-lysosomal degradation causes a comparable accumulation of APP and APP-metabolites in autophagosomes. Becn1 reduction in cell culture leads to lower levels of its binding partner Pik3c3 and increased presence of Microtubule-associated protein 1, light chain 3 (LC3. Overexpression of Becn1, on the other hand, reduces cellular APP levels. In line with these observations, we detected less BECN1 and PIK3C3 but more LC3 protein in brains of AD patients. We conclude that BECN1 regulates APP processing and turnover. BECN1 is involved in autophagy initiation and autophagosome clearance. Accordingly, BECN1 deficiency disrupts cellular autophagy and autophagosomal-lysosomal degradation and alters APP metabolism. Together, our findings suggest that autophagy and the BECN1-PIK3C3 complex regulate APP processing and play an important role in AD pathology.

  6. Transient laser spectroscopy of protein folding: detection and characterization of slow annealing processes

    Science.gov (United States)

    Subramaniam, Vinod; Bergenhem, Nils; Gafni, Ari; Steel, Duncan G.

    1995-09-01

    The unique structure of a protein is encoded in its characteristic sequence of amino acids; the processes by which this linear sequence collapses into a unique 3D structure remains an unsolved problem that represents one of the most challenging issues in fundamental biomolecular science. This so-called protein folding problem is the second half of the genetic code. Studies of this biological problem are complicated by the need to study dynamic behavior involving small populations of transient species in a solution environment. However, the use of advanced transient laser spectroscopy techniques based on intrinsic chromophores provides a powerful means to study this problem. Specifically, time-resolved phosphorescence of tryptophan (Trp) provides a means to study the dynamics associated with different regions of the protein surrounding the emitting Trp residue. Using these methodologies, we are able to study, in real time, the later stages of unfolding and refolding of the bacterial protein alkaline phosphatase, a nonspecific monoesterase. Results show the presence of several intermediate states, including states with significantly altered core structure that still exhibit complete biological activity. Moreover, the refolding of alkaline phosphatase following denaturation in either chaotropic denaturants or low pH reveals a relatively fast refolding leading to the biologically active state, while laser spectroscopy measurements show a soft core which is annealing to the native-like state on a time-scale long compared to the return of activity. The active refolded protein is also initially characterized by an increase in susceptibility to denaturant. The slow annealing of the core is consistent with the presence of high energy barriers that separate fully active, long-lived, kinetic intermediate states along the folding pathway, a description suggested in the rugged energy landscape model.

  7. 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

  8. Biological effect of Muller's Ratchet: distant capsid site can affect picornavirus protein processing.

    Science.gov (United States)

    Escarmís, Cristina; Perales, Celia; Domingo, Esteban

    2009-07-01

    Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21 cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical level the biological defects that arise when viruses are subjected to multiple bottleneck events.

  9. Effect of physical properties of nanogel particles on the kinetic constants of multipoint protein recognition process.

    Science.gov (United States)

    Nakamoto, Masahiko; Hoshino, Yu; Miura, Yoshiko

    2014-02-10

    We report the effect of physical properties, such as flexibility and polymer density, of nanogel particles (NPs) on the association/dissociation rates constant (kon and koff) and equilibrium constants (Kd) of multipoint protein recognition process. NPs having different flexibilities and densities at 25 °C were synthesized by tuning cross-linking degrees and the volume phase transition (VPT) temperature. Rate constants were quantified by analyzing time course of protein binding process on NPs monitored by a quartz crystal microbalance (QCM). Both kon and koff of swollen phase NPs increased with decreasing cross-linking degree, whereas cross-linking degree did not affect kon and koff of the collapsed phase NPs, indicating that polymer density of NPs governs kon and koff. The results also suggest that the mechanical flexibility of NPs, defined as the Young's modulus, does not always have crucial roles in the multipoint molecular recognition process. On the other hand, Kd was independent of the cross-linking degree and depended only on the phase of NPs, indicating that molecular-scale flexibility, such as side-chain and segmental-mode mobility, as well as the conformation change, of polymer chains assist the formation of stable binding sites in NPs. Our results reveal the rationale for designing NPs having desired affinity and binding kinetics to target molecules.

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

    Energy Technology Data Exchange (ETDEWEB)

    Brosey, Chris A [Vanderbilt University; Yan, Chunli [Georgia State University, Atlanta; Tsutakawa, Susan E [Lawrence Berkeley National Laboratory (LBNL); Heller, William T [ORNL; Rambo, Robert P [Lawrence Berkeley National Laboratory (LBNL); Tainer, John A [Lawrence Berkeley National Laboratory, The Scripps Research Institite and The Skaggs Institute; Ivanov, Ivaylo [Georgia State University, Atlanta; Chazin, Walter J [Vanderbilt University

    2013-01-01

    By coupling the protection and organization of ssDNA with the 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 manages to coordinate the 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. It has been long held that RPA engages ssDNA in three stages, but our data reveal that RPA undergoes two rather than three transitions as it binds ssDNA. In contrast to previous models, RPA is more compact when fully engaged on 20-30 nucleotides of ssDNA than when DNA-free, and there is no evidence for significant population of a highly compacted structure in the initial 8-10 nucleotide binding mode. These results provide a new framework for understanding the integration of ssDNA into DNA processing machinery and how binding partners may manipulate RPA architecture to gain access to the substrate.

  11. LAFIRE: software for automating the refinement process of protein-structure analysis.

    Science.gov (United States)

    Yao, Min; Zhou, Yong; Tanaka, Isao

    2006-02-01

    Manual intervention is usually required in the multiple rounds of refinement of protein crystal structures, including linking and/or extending the fragments of the initial model and rebuilding (fitting) ill-matched residues using computer-graphics software. Such manual modification is both time-consuming and requires a great deal of expertise in crystallography. Consequently, the refinement process becomes the bottleneck for high-throughput structure analysis. A program, Local correlation coefficient-based Automatic FItting for REfinement (LAFIRE), has been developed to achieve manual intervention-free refinement. This program was designed to perform the entire process of protein structural refinement automatically using the refinement programs CNS1.1 (CNS v.1.1) or REFMAC5. The automatic process begins from an initial model, which can be approximate, fragmentary or even only main-chain, and refines it to the final model including water molecules, controlled by monitoring the R(free) factor. More than 30 structures have now been refined successfully in a fully or semi-automatic manner within a few hours or days using LAFIRE.

  12. Molecular mechanisms of viral immune evasion proteins to inhibit MHC class I antigen processing and presentation.

    Science.gov (United States)

    Zhou, Fang

    2009-01-01

    Viral products inhibit MHC class I antigen processing and presentation via three major pathways: inhibition of major histocompatibility complex (MHC) class I expression on cells, blockade of peptide trafficking and loading on MHC class I molecules, and inhibition of peptide generation in host cells. Viral products also interfere with IFN-gamma -mediated JAK/STAT signal transduction in cells. These results imply that viral proteins probably inhibit the function of IFN-gamma in MHC class I antigen presentation via inactivation of JAK/STAT signal transduction in host cells. Mechanisms of viral products to inhibit IFN-gamma -mediated MHC class I antigen presentation were summarized in this literature review.

  13. Effect of drying process assisted by high-pressure impregnation on protein quality and digestibility in red abalone (Haliotis rufescens).

    Science.gov (United States)

    Cepero-Betancourt, Yamira; Oliva-Moresco, Patricio; Pasten-Contreras, Alexis; Tabilo-Munizaga, Gipsy; Pérez-Won, Mario; Moreno-Osorio, Luis; Lemus-Mondaca, Roberto

    2017-10-01

    Abalone (Haliotis spp.) is an exotic seafood product recognized as a protein source of high biological value. Traditional methods used to preserve foods such as drying technology can affect their nutritional quality (protein quality and digestibility). A 28-day rat feeding study was conducted to evaluate the effects of the drying process assisted by high-pressure impregnation (HPI) (350, 450, and 500 MPa × 5 min) on chemical proximate and amino acid compositions and nutritional parameters, such as protein efficiency ratio (PER), true digestibility (TD), net protein ratio, and protein digestibility corrected amino acid score (PDCAAS) of dried abalone. The HPI-assisted drying process ensured excellent protein quality based on PER values, regardless of the pressure level. At 350 and 500 MPa, the HPI-assisted drying process had no negative effect on TD and PDCAAS then, based on nutritional parameters analysed, we recommend HPI-assisted drying process at 350 MPa × 5 min as the best process condition to dry abalone. Variations in nutritional parameters compared to casein protein were observed; nevertheless, the high protein quality and digestibility of HPI-assisted dried abalones were maintained to satisfy the metabolic demands of human beings.

  14. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

    Science.gov (United States)

    Rocca, Jennifer D.; Hall, Edward K.; Lennon, Jay T.; Evans, Sarah E.; Waldrop, Mark P.; Cotner, James B.; Nemergut, Diana R.; Graham, Emily B.; Wallenstein, Matthew D.

    2015-01-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes.

  15. Dynamical modeling of microRNA action on the protein translation process

    Directory of Open Access Journals (Sweden)

    Barillot Emmanuel

    2010-02-01

    Full Text Available Abstract Background Protein translation is a multistep process which can be represented as a cascade of biochemical reactions (initiation, ribosome assembly, elongation, etc., the rate of which can be regulated by small non-coding microRNAs through multiple mechanisms. It remains unclear what mechanisms of microRNA action are the most dominant: moreover, many experimental reports deliver controversial messages on what is the concrete mechanism actually observed in the experiment. Nissan and Parker have recently demonstrated that it might be impossible to distinguish alternative biological hypotheses using the steady state data on the rate of protein synthesis. For their analysis they used two simple kinetic models of protein translation. Results In contrary to the study by Nissan and Parker, we show that dynamical data allow discriminating some of the mechanisms of microRNA action. We demonstrate this using the same models as developed by Nissan and Parker for the sake of comparison but the methods developed (asymptotology of biochemical networks can be used for other models. We formulate a hypothesis that the effect of microRNA action is measurable and observable only if it affects the dominant system (generalization of the limiting step notion for complex networks of the protein translation machinery. The dominant system can vary in different experimental conditions that can partially explain the existing controversy of some of the experimental data. Conclusions Our analysis of the transient protein translation dynamics shows that it gives enough information to verify or reject a hypothesis about a particular molecular mechanism of microRNA action on protein translation. For multiscale systems only that action of microRNA is distinguishable which affects the parameters of dominant system (critical parameters, or changes the dominant system itself. Dominant systems generalize and further develop the old and very popular idea of limiting step

  16. Aroma extract dilution analysis of a beeflike process flavor from extruded enzyme-hydrolyzed soybean protein.

    Science.gov (United States)

    Baek, H H; Kim, C J; Ahn, B H; Nam, H S; Cadwallader, K R

    2001-02-01

    Aroma-active compounds from a beeflike process flavor, produced by extrusion of enzyme-hydrolyzed vegetable protein (E-HVP), were analyzed using aroma extract dilution analysis. The number of aroma-active compounds and the aroma intensity were increased by the addition of aroma precursors prior to extrusion. The most intense compound was 2-methyl-3-furanthiol having a cooked rice/vitamin-like/meaty aroma note. Several sulfur-containing furans, such as 2-methyl-3-(methylthio)furan, 2-methyl-3-(methyldithio)furan, and bis(2-methylfuryl)disulfide, were detected with high flavor dilution (FD) factors. Some pyrazines, such as 2-ethyl-3,5-dimethylpyrazine, 2,6-diethylpyrazine, and 3,5-diethyl-2-methylpyrazine, also had high FD factors. It is hypothesized that sulfur-containing amino acids and thiamin were important precursors in aroma formation in process flavor from E-HVP.

  17. 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.

  18. Dietary protein and fat emulsions, processed by ultrasound and pulsed magnetic field

    Directory of Open Access Journals (Sweden)

    E. I. Verboloz

    2017-01-01

    Full Text Available For the baking of baked goods in order to save fats, different types of endorsement and protein-fatty emulsions which are used as ingredients in goods and for the protection of metal moulds from burning. Usually emulsion is prepared on bakery enterprises by National State Standard Р 51785–2001, involving mechanical beating up of ingredients. The authors suggested and studied the way of manufacturing of more stable food protein-fatty emulsions using ultrasonic transmitter with rigid neodymium magnets on its thickener. As ingredients, there were applied curd whey diluted with water, unpurified sunflower oil and sunflower phosphatides. Ratio of whey and water is 1:7. Physical effects of ultrasound and field of magnets in contact layer of liquid ingredients being dispersed have increased the viscosity and dispersion of protein-fatty emulsions. Hypothesis of increase of stability and sterility of protein-fatty emulsion by the selection of parameters of magnetic field and power of ultrasound transmitter is confirmed experimentally. Microscopic analysis shows high degree of homogeneity of emulsion under the time of processing 3-4 minutes and intensity of ultrasound 2 W/cm2, that is energetically profitable. There was revealed synergism of influence of physical effects of ultrasound and magnetic field on the durability and steadiness of emulsion to mechanical and temperature effect and also cidal effect, prolonging terms of product using. Manufacture of emulsions by the declared way using the ultrasound and magnetic field of constant neodymium magnets decreases number of injected elements-emulsifiers by 3-4 times or excludes their use at all. Existing piezoelectric ultrasound units as well as neodymium magnets have small sizes and low energy consumption, easily built into the line of continuous manufacture of emulsion for the bread production. Such emulsions are less demanding to the storage and transportation.

  19. Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

    Science.gov (United States)

    Sato, Osamu; Komatsu, Satoshi; Sakai, Tsuyoshi; Tsukasaki, Yoshikazu; Tanaka, Ryosuke; Mizutani, Takeomi; Watanabe, Tomonobu M; Ikebe, Reiko; Ikebe, Mitsuo

    2017-06-30

    Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s -1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s -1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. 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. © 2016 Müller-McNicoll et al.; Published by Cold Spring Harbor Laboratory Press.

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

    International Nuclear Information System (INIS)

    Gualdrón-López, Melisa; Michels, Paul A.M.

    2013-01-01

    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 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 r of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and 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

  2. 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

    2015-08-05

    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.

  3. Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants.

    Science.gov (United States)

    Buyel, Johannes F; Fischer, Rainer

    2014-04-01

    Plants offer a valuable alternative to cultured mammalian cells for the production of recombinant biopharmaceutical proteins. However, the target protein typically represents only a minor fraction of the total protein in the initial plant extract, which means that the development of product-specific chromatography-based purification strategies is often laborious and expensive. To address this challenge, we designed a generic downstream process that is suitable for the purification of recombinant proteins with diverse properties from plant production platforms. This was achieved by focusing on the binding behavior of tobacco host cell proteins (HCPs) to a broad set of chromatography resins under different pH and conductivity conditions. Strong cation exchanger and salt-tolerant anion exchanger resins exhibited the best resolution of tobacco HCPs among the 13 tested resins, and their selectivity was easy to manipulate through the adjustment of pH and conductivity. The advantages, such as direct capture of a target protein from leaf extract, and limitations, such as low binding capacity, of various chromatography ligands and resins are discussed. We also address the most useful applications of the chromatography ligands, namely recovery of proteins with a certain pI, in a downstream process that aims to purify diverse plant-derived biopharmaceutical proteins. Based on these results, we describe generic purification schemes that are suitable for acidic, neutral, and basic target proteins, as a first step toward the development of industrial platform processes. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. RNA processing and protein expression of HLA-B*07:44N.

    Science.gov (United States)

    Balas, A; García-Sánchez, F; Vicario, J L

    2017-04-01

    The assignment of human leukocyte antigen (HLA) null alleles is clinically relevant in the setting of stem cell transplantation. Cell surface expression profiling and mRNA processing analysis of the HLA-B allele previously designated as B*07:44, have been performed. Cell surface expression of HLA-B*07:44 was determined using flow cytometry. Genomic full-length and HLA-B*07-specific cDNA sequencing were carried out by Sanger procedure. Flow cytometric analysis confirmed previous serologic results and demonstrated a lack of cell membrane expression of the HLA-B protein. The mRNA processing, studied using direct HLA-B*07-specific cDNA sequencing, revealed the presence of a unique, aberrantly spliced mRNA, with a deletion of the last 43 bp on the 5'-end of exon 4. The substitution from T to G at genomic position 1799 compared to B*07:02:01 introduced a new and stronger splice donor site at exon 4. This alternative splicing produced an mRNA containing a premature stop codon at position 280, explaining the absence of mature HLA-B7 protein on the cell surface. These findings led us to consider this HLA-B variant as a HLA null allele. The World Health Organization (WHO) Nomenclature Committee has since renamed this variant B*07:44N . © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Therapeutic targeting of amyloid precursor protein and its processing enzymes for breast cancer treatment.

    Science.gov (United States)

    Rizvi, Syed Mohd Danish; Hussain, Talib; Subaiea, Gehad M; Shakil, Shazi; Ahmad, Adnan

    2017-08-28

    Breast cancer cases in women are increasing at an alarming rate globally and extensive research is being conducted to identify a breakthrough medicine against this dreadful disease. In fact, researchers are looking for fresh targets to develop novel treatment strategies for cancer of the breasts. In this article, 'amyloid precursor protein' or (APP) and its processing enzymes are deeply studied so as to explore the same as prospective targets for breast cancer treatment. Even though most of the studies on APP and its processing enzymes have been performed on neuronal cells owing to their linkage with Alzheimer's disease, they are omnipresent on various non-neuronal cells also. Interestingly, APP and its processing enzymes have a role in the proliferation of cancer cells as well as in their growth, adherence and movement. Over-synthesis of APP and its processing enzymes are emerging as important hallmark features in breast cancer. It has been found that APP and its processing enzymes, i.e., g-secretase and a-secretase are strongly linked with breast cancer via Akt phosporylation and Notch signaling pathways. Thus, targeting APP or g-secretase or a-secretase could be considered as an effective strategy to treat breast cancer and even metastasis. There are various clinical trials which are in progress to explore the potential of g-secretase inhibitor against breast cancer. Hence, the present review is composed of two sections, one section deals with all the possible linkages of APP and APP processing enzymes (a-secretase, b-secretase and g-secretase) with breast cancer. However, the other section provides recent information on breast cancer treatment strategy using APP and APP processing enzymes as targets. We strongly believe that compilation of these studies would be beneficial to the scientist working in the field of 'breast cancer-treatment'. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Fluoxetine requires the endfeet protein aquaporin-4 to enhance plasticity of astrocyte processes

    Directory of Open Access Journals (Sweden)

    Barbara eDi Benedetto

    2016-02-01

    Full Text Available Morphological alterations in astrocytes are characteristic for post mortem brains of patients affected by major depressive disorder (MDD. Recently, a significant reduction in the coverage of blood vessels (BVs by aquaporin-4 (AQP-4-positive astrocyte endfeet has been shown in the prefrontal cortex (PFC of MDD patients, suggesting that either alterations in the morphology of endfeet or in AQP-4 distribution might be responsible for the disease phenotype or constitute a consequence of its progress. Antidepressant drugs (ADs regulate the expression of several proteins, including astrocyte-specific ones. Thus, they may target AQP-4 to induce morphological changes in astrocytes and restore their proper shape or relocate AQP-4 to endfeet. Using an animal model of depression, rats selectively bred for high anxiety-like behavior (HAB, we confirmed a reduced coverage of BVs in the adult PFC by AQP-4-immunoreactive (AQP-4-IR astrocyte processes with respect to nonselected Wistar rats (NAB, thereby validating it for our study. A further evaluation of the morphology of astrocyte in brain slices (ex vivo and in vitro using an antibody against the astrocyte-specific cytoskeletal protein glial fibrillary acidic protein (GFAP revealed that HAB astrocytes extended less processes than NAB cells. Furthermore, short-term drug treatment in vitro with the AD fluoxetine (FLX was sufficient to increase the plasticity of astrocyte processes, enhancing their number in NAB-derived cells and recovering their basal number in HAB-derived cells. This enhanced FLX-dependent plasticity occurred, however, only in the presence of intact AQP-4, as demonstrated by the lack of effect after the downregulation of AQP-4 with RNAi in both NAB and HAB cells. Nonetheless, a similar short-term treatment did neither modulate the coverage of BVs with AQP-4-positive astrocyte endfeet in NAB nor in HAB rats, although dosage and time of treatment were sufficient to fully recover GFAP expression

  7. Profiling the effects of process changes on residual host cell proteins in biotherapeutics by mass spectrometry.

    Science.gov (United States)

    Schenauer, Matthew R; Flynn, Gregory C; Goetze, Andrew M

    2013-01-01

    An advanced liquid chromatography/mass spectrometry (MS) platform was used to identify and quantify residual Escherichia coli host cell proteins (HCPs) in the drug substance (DS) of several peptibodies (Pbs). Significantly different HCP impurity profiles were observed among different biotherapeutic Pbs as well as one Pb purified via multiple processes. The results can be rationally interpreted in terms of differences among the purification processes, and demonstrate the power of this technique to sensitively monitor both the quantity and composition of residual HCPs in DS, where these may represent a safety risk to patients. The breadth of information obtained using MS is compared to traditional multiproduct enzyme-linked immunosorbent assay (ELISA) values for total HCP in the same samples and shows that, in this case, the ELISA failed to detect multiple HCPs. The HCP composition of two upstream samples was also analyzed and used to demonstrate that HCPs that carry through purification processes to be detectable in DS are not always among those that are the most abundant upstream. Compared to ELISA, we demonstrate that MS can provide a more comprehensive, and accurate, characterization of DS HCPs, thereby facilitating process development as well as more rationally assessing potential safety risks posed by individual, identified HCPs. © 2013 American Institute of Chemical Engineers.

  8. A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E.

    Science.gov (United States)

    Ehgartner, Daniela; Sagmeister, Patrick; Langemann, Timo; Meitz, Andrea; Lubitz, Werner; Herwig, Christoph

    2017-07-01

    Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h -1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of -0.8 ± 0.3 h -1 . This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body

  9. Filling the void: a role for exercise-induced BDNF and brain amyloid precursor protein processing.

    Science.gov (United States)

    MacPherson, Rebecca E K

    2017-11-01

    Inactivity, obesity, and insulin resistance are significant risk factors for the development of Alzheimer's disease (AD). Several studies have demonstrated that diet-induced obesity, inactivity, and insulin resistance exacerbate the neuropathological hallmarks of AD. The aggregation of β-amyloid peptides is one of these hallmarks. β-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in amyloid precursor protein (APP) processing, leading to β-amyloid peptide formation. Understanding how BACE1 content and activity are regulated is essential for establishing therapies aimed at reducing and/or slowing the progression of AD. Exercise training has been proven to reduce the risk of AD as well as decrease β-amyloid production and BACE1 content and/or activity. However, these long-term interventions also result in improvements in adiposity, circulating metabolites, glucose tolerance, and insulin sensitivity making it difficult to determine the direct effects of exercise on brain APP processing. This review highlights this large void in our knowledge and discusses our current understanding of the direct of effect of exercise on β-amyloid production. We have concentrated on the central role that brain-derived neurotrophic factor (BDNF) may play in mediating the direct effects of exercise on reducing brain BACE1 content and activity as well as β-amyloid production. Future studies should aim to generate a greater understanding of how obesity and exercise can directly alter APP processing and AD-related pathologies. This knowledge could provide evidence-based hypotheses for designing therapies to reduce the risk of AD and dementia. Copyright © 2017 the American Physiological Society.

  10. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    International Nuclear Information System (INIS)

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein

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

    International Nuclear Information System (INIS)

    Farag, H.; Diaa El-Din, M.; El-Niely, H. F.G.

    2006-01-01

    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

  12. 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

    2015-09-21

    Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to 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.

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

    International Nuclear Information System (INIS)

    Noh, B.; Richardson, T.

    1989-01-01

    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 14 C-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

  14. Production and spray drying of protein hydrolyzate obtained from tilapia processing by-products

    Directory of Open Access Journals (Sweden)

    Leandro Daniel De Paris

    2016-01-01

    Full Text Available In the last few decades, the offer of by-products obtained from the processing of tilapia (Oreochromis niloticus has increased, and the need for developing products with high biological and nutritional values for use in animal nutrition motivated this study. Enzymatic hydrolysis of carcass, head and skin of tilapia was performed, as well as the separation of oil, residual solids and soluble proteins by centrifugation at high temperature and the spray drying of the protein fraction. Factorial designs were employed in the assays to evaluate the operating conditions of the spray dryer (inlet and outlet temperatures and flow rate and the inclusion of drying aid agents (maltodextrin and calcium carbonate. The spray drying showed the best results with air inlet temperature of 190ºC, outlet temperature of 90ºC, flow rate of 30 L·h-1 including 10% maltodextrin (mass in the liquid feed as a drying aid. The final powder recovery was higher than 90% and the physical, chemical and microbiological analyses met the Brazilian legal standards.

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

    Energy Technology Data Exchange (ETDEWEB)

    Noh, B.; Richardson, T. (Univ. of California, Davis (USA))

    1989-07-01

    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 {alpha}-lactalbumin or {beta}-lactoglobulin and the caseins studied using tracer amounts of added {sup 14}C-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% {beta}-lactoglobulin and less than .3% {alpha}-lactalbumin were incorporated into the curd. Heating at 95{degree}C for .5 to 20 min resulted in 58 to 85% of the {beta}-lactoglobulin and 8 to 55% of the {alpha}-lactalbumin becoming associated with the curd. Heating at 140{degree}C for 2 and 4 s caused 43 and 54% of the {beta}-lactoglobulin and 9 and 12% of the {alpha}-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.

  16. Expression of hepatitis C virus proteins does not interfere with major histocompatibility complex class I processing and presentation in vitro.

    Science.gov (United States)

    Moradpour, D; Grabscheid, B; Kammer, A R; Schmidtke, G; Groettrup, M; Blum, H E; Cerny, A

    2001-05-01

    Hepatitis C virus (HCV) infection takes a chronic course in the majority of patients. The mechanisms underlying the evasion of the host immune response and viral persistence are poorly understood. In this context, we investigated interactions of HCV proteins with major histocompatibility complex (MHC) class I processing and presentation pathways using cell lines that allow the tetracycline-regulated expression of viral structural and nonstructural proteins. These well-characterized inducible cell lines were found to efficiently process and present endogenously synthesized HCV proteins via MHC class I. Functional MHC class I cell-surface expression and intracellular proteasome activity were not affected by the expression of HCV proteins. These results suggest that viral evasion of the host immune response does not involve interactions of HCV with MHC class I processing and presentation. Other mechanisms, such as interference with the interferon system, may be operative in HCV infection, leading to viral persistence.

  17. Proteins Involved in Distinct Phases of Cold Hardening Process in Frost Resistant Winter Barley (Hordeum vulgare L. cv Luxor

    Directory of Open Access Journals (Sweden)

    Radovan Hynek

    2013-04-01

    Full Text Available Winter barley is an economically important cereal crop grown in higher latitudes and altitudes where low temperatures represent an important environmental constraint limiting crop productivity. In this study changes in proteome of leaves and crowns in a frost tolerant winter barley cv. Luxor in relation to short and long term periods of cold followed by a brief frost treatment were studied in order to disclose proteins responsible for the cold hardening process in distinct plant tissues. The mentioned changes have been monitored using two dimensional difference gel electrophoresis (2D-DIGE with subsequent peptide-mapping protein identification. Regarding approximately 600–700 distinct protein spots detected on 2D gels, there has been found at least a two-fold change after exposure to low temperatures in about 10% of proteins in leaves and 13% of proteins in crowns. Protein and nitrogen metabolic processes have been influenced by low temperature to a similar extent in both tissues while catabolism, carbohydrate metabolism and proteins involved in stress response have been more affected in crowns than in leaves. The range of changes in protein abundance was generally higher in leaves and chloroplast proteins were frequently affected which suggests a priority to protect photosynthetic apparatus. Overall, our data proved existence of slightly different response strategies to low temperature stress in crowns and leaves, i.e., tissues with different biological role. Moreover, there have been found several proteins with large increase in accumulation, e.g., 33 kDa oxygen evolving protein of photosystem II in leaves and “enhanced disease susceptibility 1” in crowns; these proteins might have potential to indicate an enhanced level of frost tolerance in barley.

  18. 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.

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

    Science.gov (United States)

    Joshi, Deepti; Soni, R. K.

    2015-05-01

    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.

  20. 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.

  1. MODELING OF THE SPRAY DRYING PROCESS OF GREEN PROTEIN SUSPENSION CONCENTRATE (PGC

    Directory of Open Access Journals (Sweden)

    A. A. Shevtsov

    2015-01-01

    Full Text Available Development and implementation of high-tech and energy-efficient methods of feed production is important and ap¬propriate due to the fact that enterprises are not able to provide the market of feed consumers with high quality products at affordable prices. To solve this problem, an alternative technology for the production of protein green concentrate (PGC from the cormophyte mass of high protein plants was developed. The most energy-intensive process of obtaining PGC is spray drying. At the same time the problems of energy saving, and the product quality are solved by modeling. The drying model developed in this study is based on the falling edge of evaporation, which is used in many studies of drops drying. The problem of obtaining the basic equations of heat and mass transfer during the periods of constant and decreasing drying rate was to be solved. It is also supposed that the drying takes place during the periods of constant and decreasing drying rate. Basic equations of heat and mass transfer for both periods of drying were obtained. Changing of thermophysical characteristics were determined by statistical methods in the range of PGC humidity of 10 ... 75% and a temperature of 20 ... 100%. The model is solved by finite difference method with an accuracy of modeling results of 12%. Method of finite differences is a numerical method for solving differential equations based on the replacement of derivative differences schemes and is the grid method. Identification of model parameters to experimental data obtained in the experimental spray dryer was carried out. The solution allows the mathematical model to determine the change in moisture content (DS concentration and drop radial temperature in the spray drying of the PGC concentrate that is necessary both to select the geometrical sizes of the dryer and the drying process parameters controlling.

  2. Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

    Directory of Open Access Journals (Sweden)

    Rotwein Peter

    2008-04-01

    Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

  3. The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process.

    Science.gov (United States)

    Hogwood, Catherine E M; Tait, Andrew S; Koloteva-Levine, Nadejda; Bracewell, Daniel G; Smales, C Mark

    2013-01-01

    Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs. Copyright © 2012 Wiley Periodicals, Inc.

  4. Suspension culture process of MethA tumor cell for the production of heat-shock protein glycoprotein 96: process optimization in spinner flasks.

    Science.gov (United States)

    Tang, Ya-Jie; Li, Hong-Mei; Hamel, Jean-François P

    2007-01-01

    Heat-shock proteins (HSPs) act like "chaperones", making sure that the cell's proteins are in the right shape and in the right place at the right time. Heat-shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat-shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat-shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat-shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat-shock protein gp96. Initial glucose concentration had a significant effect on the heat-shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat-shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat-shock protein gp96

  5. 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

  6. A three-part signal governs differential processing of Gli1 and Gli3 proteins by the proteasome.

    Science.gov (United States)

    Schrader, Erin K; Harstad, Kristine G; Holmgren, Robert A; Matouschek, Andreas

    2011-11-11

    The Gli proteins are the transcriptional effectors of the mammalian Hedgehog signaling pathway. In an unusual mechanism, the proteasome partially degrades or processes Gli3 in the absence of Hedgehog pathway stimulation to create a Gli3 fragment that opposes the activity of the full-length protein. In contrast, Gli1 is not processed but degraded completely, despite considerable homology with Gli3. We found that these differences in processing can be described by defining a processing signal that is composed of three parts: the zinc finger domain, an adjacent linker sequence, and a degron. Gli3 processing is inhibited when any one component of the processing signal is disrupted. We show that the zinc fingers are required for processing only as a folded structure and that the location but not the identity of the processing degron is critical. Within the linker sequence, regions of low sequence complexity play a crucial role, but other sequence features are also important. Gli1 is not processed because two components of the processing signal, the linker sequence and the degron, are ineffective. These findings provide new insights into the molecular elements that regulate Gli protein processing by the proteasome.

  7. A Three-part Signal Governs Differential Processing of Gli1 and Gli3 Proteins by the Proteasome*

    Science.gov (United States)

    Schrader, Erin K.; Harstad, Kristine G.; Holmgren, Robert A.; Matouschek, Andreas

    2011-01-01

    The Gli proteins are the transcriptional effectors of the mammalian Hedgehog signaling pathway. In an unusual mechanism, the proteasome partially degrades or processes Gli3 in the absence of Hedgehog pathway stimulation to create a Gli3 fragment that opposes the activity of the full-length protein. In contrast, Gli1 is not processed but degraded completely, despite considerable homology with Gli3. We found that these differences in processing can be described by defining a processing signal that is composed of three parts: the zinc finger domain, an adjacent linker sequence, and a degron. Gli3 processing is inhibited when any one component of the processing signal is disrupted. We show that the zinc fingers are required for processing only as a folded structure and that the location but not the identity of the processing degron is critical. Within the linker sequence, regions of low sequence complexity play a crucial role, but other sequence features are also important. Gli1 is not processed because two components of the processing signal, the linker sequence and the degron, are ineffective. These findings provide new insights into the molecular elements that regulate Gli protein processing by the proteasome. PMID:21921029

  8. Physicochemical and functional properties of protein concentrate from by-product of coconut processing.

    Science.gov (United States)

    Rodsamran, Pattrathip; Sothornvit, Rungsinee

    2018-02-15

    Coconut cake, a by-product from milk and oil extractions, contains a high amount of protein. Protein extraction from coconut milk cake and coconut oil cake was investigated. The supernatant and precipitate protein powders from both coconut milk and oil cakes were compared based on their physicochemical and functional properties. Glutelin was the predominant protein fraction in both coconut cakes. Protein powders from milk cake presented higher water and oil absorption capacities than those from oil cake. Both protein powders from oil cake exhibited better foaming capacity and a better emulsifying activity index than those from milk cake. Coconut proteins were mostly solubilized in strong acidic and alkaline solutions. Minimum solubility was observed at pH 4, confirming the isoelectric point of coconut protein. Therefore, the coconut residues after extractions might be a potential alternative renewable plant protein source to use asa food ingredient to enhance food nutrition and quality. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Influence of cooking process on protein fractions in cooked ham and mortadella

    Directory of Open Access Journals (Sweden)

    G. Vonghia

    2011-03-01

    Full Text Available The mortadella is a pork meat sausage (in natural or artificial bowel accurately triturated and mixed with little backfat cubes, salt, sodium nitrate and nitrite, spices and peppercorns, and then cooked in oven for many hours. The cooked ham is obtained from an anatomically completed piece of meat; the working process provides the addiction of salt and spices, the brine, the bones removal, the churning and the pressing, so the cured meat is first packed in a mould provided for this purpose, then cooked and after cooled and packed. The meat cooking is the last step in the cooked sausage production technology, and let us obtain a stable and eatable product. The effect of the heat and the lenght of processing are the main responsibles for modifications in water- and salt-soluble protein fractions. Indeed myofibrils denature themselves after cooking and consequently their solubility decreases; particularly the denaturation begins over 30°C in the myosin chain, instead the actin solubility begins to decrease over 60°C, being the actin more stable than myosin (Barbieri et al., 1997...

  10. Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's β-secretase

    Science.gov (United States)

    Scholefield, Zoe; Yates, Edwin A.; Wayne, Gareth; Amour, Augustin; McDowell, William; Turnbull, Jeremy E.

    2003-01-01

    Cleavage of amyloid precursor protein (APP) by the Alzheimer's β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing. PMID:14530380

  11. 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

    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...... 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...... 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...

  12. The cost of expression of Escherichia coli lac operon proteins is in the process, not in the products.

    Science.gov (United States)

    Stoebel, Daniel M; Dean, Antony M; Dykhuizen, Daniel E

    2008-03-01

    Transcriptional regulatory networks allow bacteria to express proteins only when they are needed. Adaptive hypotheses explaining the evolution of regulatory networks assume that unneeded expression is costly and therefore decreases fitness, but the proximate cause of this cost is not clear. We show that the cost in fitness to Escherichia coli strains constitutively expressing the lactose operon when lactose is absent is associated with the process of making the lac gene products, i.e., associated with the acts of transcription and/or translation. These results reject the hypotheses that regulation exists to prevent the waste of amino acids in useless protein or the detrimental activity of unnecessary proteins. While the cost of the process of protein expression occurs in all of the environments that we tested, the expression of the lactose permease could be costly or beneficial, depending on the environment. Our results identify the basis of a single selective pressure likely acting across the entire E. coli transcriptome.

  13. 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

  14. Freeze-Drying Above the Glass Transition Temperature in Amorphous Protein Formulations While Maintaining Product Quality and Improving Process Efficiency.

    Science.gov (United States)

    Depaz, Roberto A; Pansare, Swapnil; Patel, Sajal Manubhai

    2016-01-01

    This study explored the ability to conduct primary drying during lyophilization at product temperatures above the glass transition temperature of the maximally freeze-concentrated solution (Tg′) in amorphous formulations for four proteins from three different classes. Drying above Tg′ resulted in significant reductions in lyophilization cycle time. At higher protein concentrations, formulations freeze dried above Tg′ but below the collapse temperature yielded pharmaceutically acceptable cakes. However, using an immunoglobulin G type 4 monoclonal antibody as an example, we found that as protein concentration decreased, minor extents of collapse were observed in formulations dried at higher temperatures. No other impacts to product quality, physical stability, or chemical stability were observed in this study among the different drying conditions for the different proteins. Drying amorphous formulations above Tg′, particularly high protein concentration formulations, is a viable means to achieve significant time and cost savings in freeze-drying processes.

  15. The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

    Science.gov (United States)

    Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

    2016-11-01

    The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (ppain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain perception. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Variances in nutrient content and yield of alfalfa protein concentrate processed with five methods

    Science.gov (United States)

    The demand for protein is growing with increased populations and world affluence. A sustainable and affordable protein source is needed to support the growing aquaculture industry worldwide. Alfalfa produces high levels of protein and provides numerous environmental services, potentially making it a...

  17. Effect of four processed animal proteins in the diet on digestibility and performance in laying hens.

    Science.gov (United States)

    van Krimpen, M M; Veldkamp, T; Binnendijk, G P; de Veer, R

    2010-12-01

    An experiment was performed to investigate the effect of animal vs. vegetable protein sources in the diet of laying hens on the development of hen performance. A diet containing protein sources of only vegetable origin was compared with 4 diets, each containing 1 of 4 processed animal proteins (PAP). Two PAP (Daka-58 and Sonac-60) were classified as meat meals, and the remaining 2 (Daka-40 and Sonac-50) were classified as meat and bone meals. First, fecal digestibility of nutrients in the PAP was determined in Lohmann Brown layers. Hens (n = 132) were housed in 22 cages (6 hens/cage) and allotted to 5 dietary treatments. In the PAP diets (4 replicates/treatment), 100 g/kg of CP of animal origin was added, replacing soybean meal and corn (Zea mays) in the basal diet (6 replicates/treatment). The PAP sources differed largely in chemical composition and digestibility coefficients. Energy content (AME(n)) varied from 1,817 (Daka-40) to 3,107 kcal/kg (Sonac-60), and digestible lysine varied from 15.4 (Daka-40) to 28.3 g/kg (Sonac-50). Subsequently, the effect of a control diet (without PAP) vs. 4 PAP diets (50 g/kg of CP of animal origin from the same batches as used in the digestibility study) on performance was determined. All diets were isocaloric (AME(n) = 2,825 kcal/kg) and isonitrogenous (digestible lysine = 6.8 g/kg). Hens were housed in 40 floor pens (12 hens/pen, 8 pens/treatment) from 20 to 40 wk of age. Feed intake levels of the hens fed the meat and bone meal diets were reduced compared with those of hens fed the meat meal diets, whereas the feed intake level of hens fed the control diet was intermediate. Laying hen performance differed between treatments, being was most favorable for the Sonac-50 treatment and most adverse for the Daka-40 treatment. Differences in laying hen performance seemed to be related partly to differences in feed intake and corresponding amino acid intake.

  18. A juice extractor can simplify the downstream processing of plant-derived biopharmaceutical proteins compared to blade-based homogenizers

    OpenAIRE

    Buyel, J.F.; Fischer, R.

    2015-01-01

    The production of biopharmaceutical proteins using plant-based systems has recently become economically competitive with conventional expression platforms based on microbes and mammalian cells, but downstream processing remains a significant cost factor. Here we report that, depending on the protein expression level, production costs for biopharmaceuticals made in plants can be reduced by up to 30% if a juice extractor is used instead of a blade-based homogenizer or blender. Although the extr...

  19. Expression of hepatitis C virus proteins does not interfere with major histocompatibility complex class I processing and presentation in vitro

    OpenAIRE

    Moradpour, Darius; Grabscheid, Benno; Kammer, Andreas R.; Schmidtke, Gunter; Gröttrup, Marcus; Blum, Hubert E.; Cerny, Andreas

    2001-01-01

    Hepatitis C virus (HCV) infection takes a chronic course in the majority of patients. The mechanisms underlying the evasion of the host immune response and viral persistence are poorly understood. In this context, we investigated interactions of HCV proteins with major histocompatibility complex (MHC) class I processing and presentation pathways using cell lines that allow the tetracycline-regulated expression of viral structural and nonstructural proteins. These well-characterized inducible ...

  20. Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Pager, Cara Theresia; Wurth, Mark Allen; Dutch, Rebecca Ellis

    2004-01-01

    Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK↓L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero ...

  1. A human protein interaction network shows conservation of aging processes between human and invertebrate species.

    Directory of Open Access Journals (Sweden)

    Russell Bell

    2009-03-01

    Full Text Available We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

  2. 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.

  3. Effects of thermal processing on the nutritional and functional properties of defatted conophor nut (Tetracarpidium conophorum) flour and protein isolates.

    Science.gov (United States)

    Iyenagbe, David O; Malomo, Sunday A; Idowu, Atinuke O; Badejo, Adebanjo A; Fagbemi, Tayo N

    2017-11-01

    Conophor nut ( Tetracarpidium conophorum ) was processed using different heat treatments to explore its full potential as food ingredients. The raw, boiled, and toasted nuts were defatted and the proteins isolated by alkaline solubilization and isoelectric precipitation. The variously processed nuts were analyzed for the proximate and amino acid compositions, and functional properties. The protein contents of the isolate ranges between 86.86 g/100g and 87.74 g/100 g, about 1.5-fold higher than those of the defatted flour samples. The essential amino acids of the isolates ranged between 40.57%-41.55%. Glutamic acid, aspartic acid, and arginine were the most predominant amino acids, while methionine and lysine were the first and second limiting amino acids, respectively. The protein efficiency ratio, biological values as well as the functional properties of the proteins were improved with processing. These properties may enhance the potential use of conophor nut protein isolates as high-quality protein ingredient in food systems.

  4. 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.

  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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. 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.

  7. Effect of curing agents on the oxidative and nitrosative damage to meat proteins during processing of fermented sausages.

    Science.gov (United States)

    Villaverde, A; Morcuende, D; Estévez, M

    2014-07-01

    The effect of increasing concentrations of curing agents, ascorbate (0, 250, and 500 ppm), and nitrite (0, 75, and 150 ppm), on the oxidative and nitrosative damage to proteins during processing of fermented sausages was studied. The potential influence of these reactions on color and texture of the fermented sausages was also addressed. Nitrite had a pro-oxidant effect on tryptophan depletion and promoted the formation of protein carbonyls and Schiff bases. The nitration degree in the fermented sausages was also dependent on nitrite concentration. On the other hand, ascorbate acted as an efficient inhibitor of the oxidative and nitrosative damage to meat proteins. As expected, nitrite clearly favored the formation of the cured red color and ascorbate acted as an enhancer of color formation. Nitrite content was positively correlated with hardness. The chemistry behind the action of nitrite and ascorbate on muscle proteins during meat fermentation is thoroughly discussed. The results suggest that ascorbate (500 ppm) may be required to compensate the pro-oxidant impact of nitrite on meat proteins. This study provides insight on the action of curing agents on meat proteins during processing of fermented sausages. This chemistry background provides understanding of the potential influence of the oxidative and nitrosative damage to proteins on the quality of processed muscle foods. The study provides novel information on the impact of the combination of nitrite and ascorbate on the chemical deterioration of proteins and the influence on particular quality traits of fermented sausages. These data may be of interest for the design of cured muscle foods of enhanced quality. © 2014 Institute of Food Technologists®

  8. Production of coconut protein powder from coconut wet processing waste and its characterization.

    Science.gov (United States)

    Naik, Aduja; Raghavendra, S N; Raghavarao, K S M S

    2012-07-01

    Virgin coconut oil (VCO) has been gaining popularity in recent times. During its production, byproducts such as coconut skim milk and insoluble protein are obtained which are underutilized or thrown away to the environment at present. This study deals with utilization of these byproducts to obtain a value-added product, namely, coconut protein powder. When coconut milk was subjected to centrifugation, three phases, namely, fat phase (coconut cream), aqueous phase (coconut skim milk), and solid phase (insoluble protein) were obtained. The coconut skim milk and insoluble protein were mixed and homogenized before spray drying to obtain a dehydrated protein powder. The proximate analysis of the powder showed high protein content (33 % w/w) and low fat content (3 % w/w). Protein solubility was studied as a function of pH and ionic content of solvent. Functional properties such as water hydration capacity, fat absorption capacity, emulsifying properties, wettability, and dispersibility of coconut protein powder were evaluated along with morphological characterization, polyphenol content, and color analysis. Coconut protein powder has shown to have good emulsifying properties and hence has potential to find applications in emulsified foods. Sensory analysis showed high overall quality of the product, indicating that coconut protein powder could be a useful food ingredient.

  9. Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

    Science.gov (United States)

    Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

    2018-01-01

    Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

  10. 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.

  11. Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

    2012-01-01

    Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...... response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-¿ release, and antibody induction. Gal...

  12. Interactions between milk protein ingredients and other milk components during processing

    DEFF Research Database (Denmark)

    Liu, Guanchen

    Microparticulated whey protein (MWP) are colloidal particles usually formed by combined heating and shearing of whey protein concentrates (WPC), and typically have particle sizes ranging from 1.0 to 10 μm. Nanoparticulated whey protein (NWP) have a smaller particle size (100 to 990 nm). Previous...... research in our group shown that, both MWP and NWP can give a higher viscosity and denser microstructure compared to WPC when used as fat replacer in low-fat yoghurt. In the thesis, we investigated how these two types of commercial whey protein particles interact with other milk components and how...

  13. Proteins and Amino Acids in Fine Particulate Matter in Rural Guangzhou, Southern China: Seasonal Cycles, Sources, and Atmospheric Processes.

    Science.gov (United States)

    Song, Tianli; Wang, Shan; Zhang, Yingyi; Song, Junwei; Liu, Fobang; Fu, Pingqing; Shiraiwa, Manabu; Xie, Zhiyong; Yue, Dingli; Zhong, Liuju; Zheng, Junyu; Lai, Senchao

    2017-06-20

    Water-soluble proteinaceous matter including proteins and free amino acids (FAAs) as well as some other chemical components was analyzed in fine particulate matter (PM 2.5 ) samples collected over a period of one year in rural Guangzhou. Annual averaged protein and total FAAs concentrations were 0.79 ± 0.47 μg m -3 and 0.13 ± 0.05 μg m -3 , accounting for 1.9 ± 0.7% and 0.3 ± 0.1% of PM 2.5 , respectively. Among FAAs, glycine was the most abundant species (19.9%), followed by valine (18.5%), methionine (16.1%), and phenylalanine (13.5%). Both proteins and FAAs exhibited distinct seasonal variations with higher concentrations in autumn and winter than those in spring and summer. Correlation analysis suggests that aerosol proteinaceous matter was mainly derived from intensive agricultural activities, biomass burning, and fugitive dust/soil resuspension. Significant correlations between proteins/FAAs and atmospheric oxidant (O 3 ) indicate that proteins/FAAs may be involved in O 3 related atmospheric processes. Our observation confirms that ambient FAAs could be degraded from proteins under the influence of O 3 , and the stoichiometric coefficients of the reactions were estimated for FAAs and glycine. This finding provides a possible pathway for the production of aerosol FAAs in the atmosphere, which will improve the current understanding on atmospheric processes of proteinaceous matter.

  14. Crystallization Process of Protein Rv0731c from Mycobacterium Tuberculosis for a Successful Atomic Resolution Crystal Structure at 1.2 Angstrom

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang Cong

    2009-06-08

    Proteins are bio-macromolecules consisting of basic 20 amino acids and have distinct three-dimensional folds. They are essential parts of organisms and participate in every process within cells. Proteins are crucial for human life, and each protein within the body has a specific function, such as antibodies, contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins and transport proteins. Determining three-dimensional structure of a protein can help researchers discover the remarkable protein folding, binding site, conformation and etc, in order to understand well of protein interaction and aid for possible drug design. The research on protein structure by X-ray protein crystallography carried by Li-Wei Hung's research group in the Physical Bioscience Division at Lawrence Berkeley National Laboratory (LBNL) is focusing on protein crystallography. The research in this lab is in the process of from crystallizing the proteins to determining the three dimensional crystal structures of proteins. Most protein targets are selected from Mycobacterium Tuberculosis. TB (Tuberculosis) is a possible fatal infectious disease. By studying TB target protein can help discover antituberculer drugs, and find treatment for TB. The high-throughput mode of crystallization, crystal harvesting, crystal screening and data collection are applied to the research pipeline (Figure 1). The X-ray diffraction data by protein crystals can be processed and analyzed to result in a three dimensional representation of electron density, producing a detailed model of protein structure. Rv0731c is a conserved hypothetical protein with unknown function from Mycobacterium Tuberculosis. This paper is going to report the crystallization process and brief structure information of Rv0731c.

  15. Improvement of the digestibility of the proteins of the red alga Palmaria palmata by physical processes and fermentation.

    Science.gov (United States)

    Marrion, Olivier; Schwertz, Annie; Fleurence, Joël; Guéant, Jean Louis; Villaume, Christian

    2003-10-01

    Palmaria palmata (dulse) is an edible red alga constituting a potential protein source in human diet. However, previous studies showed that the digestibility of dulse proteins is bad because of the cell-wall encapsulating cytoplasmic proteins and the presence of fibers. The water-soluble xylan, present in high proportions in dulse, could be involved to explain the weak digestibility of proteins. To limit the influence of fibers and to improve the nutritional quality of these proteins, we have treated dulse by physical processes or by fermentation by moulds. After a 30 min predigestion by pepsin followed by a 6 h digestion into a cell dialysis containing porcine pancreatin, the corrected in vitro digestibility of crude dulse was very low (about 1.5% after correction by digestibility blank). The in vitro protein digestibility was estimated to 58% of that of casein for dulse samples obtained after washing in demineralized water and grinding in liquid nitrogen. The in vitro protein digestibility of fermented samples was 45%-65% of that of casein. After physical treatment, the digestibility improvement was related to the elimination of soluble molecules such as xylan and mineral salts. The improvement observed after fermentations seemed due to the degradation of insoluble fibers.

  16. 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.

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

    Science.gov (United States)

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

    2015-07-01

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

  18. 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

    2015-04-21

    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.

  19. 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

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

    Directory of Open Access Journals (Sweden)

    Chiara Mozzetti Monterumici

    2015-04-01

    Full Text Available 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.

  1. Nutritional value and digestion rate of rhea meat proteins in association with storage and cooking processes.

    Science.gov (United States)

    Filgueras, Renata S; Gatellier, Philippe; Ferreira, Claude; Zambiazi, Rui C; Santé-Lhoutellier, Véronique

    2011-09-01

    The nutritional value of proteins was investigated after the storage and cooking of rhea M. Gastrocnemius pars interna. Oxidation of basic and aromatic amino acids, surface hydrophobicity and aggregation state of proteins, were determined in raw and cooked meat. In addition, myofibrillar proteins were exposed in vitro to proteases of the digestive tract. Cooking markedly affected the protein surface hydrophobicity. The BBP bound content was three times greater in cooked than in fresh rhea meat. A small increment in tryptophan content after cooking was observed. Storage influenced Schiff bases formation indicating the presence of protein-aldehyde adducts after cooking. High content of Schiff bases was found after cooking of samples stored for 5 days, demonstrating a probable implication of free amino groups, most likely from lysine. Cooking decreased the myofibrillar protein susceptibility to pepsin activity. After cooking, the proteolysis rate by pancreatic enzymes increased. Our findings support the importance of protein aggregation in the nutritional value of meat proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Role of Ser-Arg Proteins in the Regulation of RNA Processing

    National Research Council Canada - National Science Library

    Blencowe, Benjamin J

    1999-01-01

    We have identified a complex SR-related matrix proteins of 16OkDa and 3OOkDa (SRm16O/3OO) that functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins...

  3. Development of a protein-rich composite sorghum-cowpea instant porridge by extrusion cooking process

    CSIR Research Space (South Africa)

    Pelembe, LAM

    2002-01-01

    Full Text Available To develop instant high protein porridge, various ratios of sorghum and cowpeas were extruded at 130 and 165 degrees C and a water content of 200 g/kg using a thin-screw extruder. An increased proportion of cowpeas resulted in an increase in protein...

  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

    2016-11-05

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Cancer associated aberrant protein O-glycosylation can modify antigen processing and immune response.

    Directory of Open Access Journals (Sweden)

    Caroline B Madsen

    Full Text Available Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA-MUC1 fusion peptides (+/- glycosylation loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB-Tg(HLA-A/H2-D2Enge/J (HLA-A2 transgenic mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.

  6. TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression.

    Science.gov (United States)

    Suzuki, Hidefumi; Isogai, Momoko; Maeda, Ryo; Ura, Kiyoe; Tamura, Taka-Aki

    2015-07-27

    TBP-TFIIA interaction is involved in the potentiation of TATA box-driven promoters. TFIIA activates transcription through stabilization of TATA box-bound TBP. The precursor of TFIIA is subjected to Taspase1-directed processing to generate α and β subunits. Although this processing has been assumed to be required for the promoter activation function of TFIIA, little is known about how the processing is regulated. In this study, we found that TBP-like protein (TLP), which has the highest affinity to TFIIA among known proteins, affects Taspase1-driven processing of TFIIA. TLP interfered with TFIIA processing in vivo and in vitro, and direct binding of TLP to TFIIA was essential for inhibition of the processing. We also showed that TATA box promoters are specifically potentiated by processed TFIIA. Processed TFIIA, but not unprocessed TFIIA, associated with the TATA box. In a TLP-knocked-down condition, not only the amounts of TATA box-bound TFIIA but also those of chromatin-bound TBP were significantly increased, resulting in the stimulation of TATA box-mediated gene expression. Consequently, we suggest that TLP works as a negative regulator of the TFIIA processing and represses TFIIA-governed and TATA-dependent gene expression through preventing TFIIA maturation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode.

    Science.gov (United States)

    Shamashkin, Michael; Godavarti, Ranga; Iskra, Timothy; Coffman, Jon

    2013-10-01

    A significant consequence of scaling up production of high titer monoclonal antibody (mAb) processes in existing facilities is the generation of in-process pools that exceed the capacity of storage vessels. A semi-continuous downstream process where columns and filters are linked and operated in tandem would eliminate the need for intermediate holding tanks. This study is a bench-scale demonstration of the feasibility of a tandem process for the purification of mAbs employing an affinity Protein A capture step, followed by a flow-through anion-exchange (AEX) step with the possibility of adding an in-line virus filtration step (VF). All three steps were linked sequentially and operated as one continuous process using an ÄKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX column was operated in a weak partitioning (WP) mode enabled by a precise in-line titration of Protein A effluent. In order to avoid complex control schemes and facilitate validation, quality and robustness were built into the system through selection of buffers based on thermodynamic and empirical models. The tandem system utilized the simplest possible combination of valves, pumps, controls, and automation, so that it could easily be implemented in a clinical or commercial production facility. Linking the purification steps in a tandem process is expected to generate savings in time and production costs and also reduce the size of quality systems due to reduced documentation requirements, microbial sampling, and elimination of hold time validation. Copyright © 2013 Wiley Periodicals, Inc.

  8. Analysis of different thermal processing methods of foodstuffs to optimize protein, calcium, and phosphorus content for dialysis patients.

    Science.gov (United States)

    Vrdoljak, Ivica; Panjkota Krbavčić, Ines; Bituh, Martina; Vrdoljak, Tea; Dujmić, Zoran

    2015-05-01

    To analyze how different thermal processing methods affect the protein, calcium, and phosphorus content of hospital food served to dialysis patients and to generate recommendations for preparing menus that optimize nutritional content while minimizing the risk of hyperphosphatemia. Standard Official Methods of Analysis (AOAC) methods were used to determine dry matter, protein, calcium, and phosphorus content in potatoes, fresh and frozen carrots, frozen green beans, chicken, beef and pork, frozen hake, pasta, and rice. These levels were determined both before and after boiling in water, steaming, stewing in oil or water, or roasting. Most of the thermal processing methods did not significantly reduce protein content. Boiling increased calcium content in all foodstuffs because of calcium absorption from the hard water. In contrast, stewing in oil containing a small amount of water decreased the calcium content of vegetables by 8% to 35% and of chicken meat by 12% to 40% on a dry weight basis. Some types of thermal processing significantly reduced the phosphorus content of the various foodstuffs, with levels decreasing by 27% to 43% for fresh and frozen vegetables, 10% to 49% for meat, 7% for pasta, and 22.8% for rice on a dry weight basis. On the basis of these results, we modified the thermal processing methods used to prepare a standard hospital menu for dialysis patients. Foodstuffs prepared according to the optimized menu were similar in protein content, higher in calcium, and significantly lower in phosphorus than foodstuffs prepared according to the standard menu. Boiling in water and stewing in oil containing some water significantly reduced phosphorus content without affecting protein content. Soaking meat in cold water for 1 h before thermal processing reduced phosphorus content even more. These results may help optimize the design of menus for dialysis patients. Copyright © 2015 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights

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

    Directory of Open Access Journals (Sweden)

    Mohammad Soltani

    2016-08-01

    Full Text Available 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.

  10. Azotobacter vinelandii metal storage protein: "classical" inorganic chemistry involved in Mo/W uptake and release processes.

    Science.gov (United States)

    Schemberg, Jörg; Schneider, Klaus; Fenske, Dirk; Müller, Achim

    2008-03-03

    The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12 degrees C revealed that three different steps can be distinguished for the Mo-release process. A proportion of approximately 15% at pH 6.8-7.0, an additional 25% at pH 7.2-7.5 and ca. 50% (up to 90% in total) at pH 7.6-7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30 degrees C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play-in addition to the pH value-an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110-115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12 degrees C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but

  11. 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.

  12. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    Science.gov (United States)

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pH

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

    International Nuclear Information System (INIS)

    Hoenen, Antje; Gillespie, Leah; Morgan, Garry; Heide, Peter van der; Khromykh, Alexander; Mackenzie, Jason

    2014-01-01

    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 KUN ) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNV 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 KUN under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNV 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 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

  14. Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing.

    Science.gov (United States)

    Pierre, Sandra; Maeurer, Christian; Coste, Ovidiu; Becker, Wiebke; Schmidtko, Achim; Holland, Sabrina; Wittpoth, Claus; Geisslinger, Gerd; Scholich, Klaus

    2008-12-01

    Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

  15. Preparation and characterization of protein isolate from Yellowfin tuna Thunnus albacares roe by isoelectric solubilization/precipitation process

    Directory of Open Access Journals (Sweden)

    Hyun Ji Lee

    2016-05-01

    Full Text Available Abstract Isoelectric solubilization/precipitation (ISP processing allows selective, pH-induced water solubility of proteins with concurrent separation of lipids and removal of materials not intended for human consumption such as bone, scales, skin, etc. Recovered proteins retain functional properties and nutritional value. Four roe protein isolates (RPIs from yellowfin tuna roe were prepared under different solubilization and precipitation condition (pH 11/4.5, pH 11/5.5, pH 12/4.5 and pH 12/5.5. RPIs contained 2.3–5.0 % moisture, 79.1–87.8 % protein, 5.6–7.4 % lipid and 3.0–3.8 % ash. Protein content of RPI-1 and RPI-2 precipitated at pH 4.5 and 5.5 after alkaline solubilization at pH 11, was higher than those of RPI-3 and RPI-4 after alkaline solubilization at pH 12 (P < 0.05. Lipid content (5.6–7.4 % of RPIs was lower than that of freeze-dried concentrate (10.6 %. And leucine and lysine of RPIs were the most abundant amino acids (8.8–9.4 and 8.5–8.9 g/100 g protein, respectively. S, Na, P, K as minerals were the major elements in RPIs. SDS-PAGE of RPIs showed bands at 100, 45, 25 and 15 K. Moisture and protein contents of process water as a 2’nd byproduct were 98.9–99.0 and 1.3–1.8 %, respectively. Therefore, yellowfin tuna roe isolate could be a promising source of valuable nutrients for human food and animal feeds.

  16. Effect of Processing on the in Vitro and in Vivo Protein Quality of Yellow and Green Split Peas (Pisum sativum).

    Science.gov (United States)

    Nosworthy, Matthew G; Franczyk, Adam J; Medina, Gerardo; Neufeld, Jason; Appah, Paulyn; Utioh, Alphonsus; Frohlich, Peter; House, James D

    2017-09-06

    In order to determine the effect of extrusion, baking, and cooking on the protein quality of yellow and green split peas, a rodent bioassay was conducted and compared to an in vitro method of protein quality determination. The Protein Digestibility-Corrected Amino Acid Score (PDCAAS) of green split peas (71.4%) was higher than that of yellow split peas (67.8%), on average. Similarly, the average Digestible Indispensable Amino Acid Score (DIAAS) of green split peas (69%) was higher than that of yellow split peas (67%). Cooked green pea flour had lower PDCAAS and DIAAS values (69.19% and 67%) than either extruded (73.61%, 70%) or baked (75.22%, 70%). Conversely, cooked yellow split peas had the highest PDCCAS value (69.19%), while extruded yellow split peas had the highest DIAAS value (67%). Interestingly, a strong correlation was found between in vivo and in vitro analysis of protein quality (R 2 = 0.9745). This work highlights the differences between processing methods on pea protein quality and suggests that in vitro measurements of protein digestibility could be used as a surrogate for in vivo analysis.

  17. Continuous bind-and-elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation.

    Science.gov (United States)

    Kaltenbrunner, Oliver; Diaz, Luis; Hu, Xiaochun; Shearer, Michael

    2016-07-08

    Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938-948, 2016. © 2016 American Institute of Chemical Engineers.

  18. The Impact of Capsid Proteins on Virus Removal and Inactivation During Water Treatment Processes.

    Science.gov (United States)

    Mayer, Brooke K; Yang, Yu; Gerrity, Daniel W; Abbaszadegan, Morteza

    2015-01-01

    This study examined the effect of the amino acid composition of protein capsids on virus inactivation using ultraviolet (UV) irradiation and titanium dioxide photocatalysis, and physical removal via enhanced coagulation using ferric chloride. Although genomic damage is likely more extensive than protein damage for viruses treated using UV, proteins are still substantially degraded. All amino acids demonstrated significant correlations with UV susceptibility. The hydroxyl radicals produced during photocatalysis are considered nonspecific, but they likely cause greater overall damage to virus capsid proteins relative to the genome. Oxidizing chemicals, including hydroxyl radicals, preferentially degrade amino acids over nucleotides, and the amino acid tyrosine appears to strongly influence virus inactivation. Capsid composition did not correlate strongly to virus removal during physicochemical treatment, nor did virus size. Isoelectric point may play a role in virus removal, but additional factors are likely to contribute.

  19. 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: 2.076, year: 2015

  20. Monoclonal Antibodies Production Platforms: An Opportunity Study of a Non-Protein-A Chromatographic Platform Based on Process Economics.

    Science.gov (United States)

    Grilo, António L; Mateus, Marília; Aires-Barros, Maria R; Azevedo, Ana M

    2017-12-01

    Monoclonal antibodies currently dominate the biopharmaceutical market with growing sales having reached 80 billion USD in 2016. As most top-selling mAbs are approaching the end of their patent life, biopharmaceutical companies compete fiercely in the biosimilars market. These two factors present a strong motivation for alternative process strategies and process optimization. In this work a novel purification strategy for monoclonal antibodies comprising phenylboronic acid multimodal chromatography for capture followed by polishing by ion-exchange monolithic chromatography and packed bed hydrophobic interaction chromatography is presented and compared to the traditional protein-A-based process. Although the capital investment is similar for both processes, the operation cost is 20% lower for the novel strategy. This study shows that the new process is worthwhile investing in and could present a viable alternative to the platform process used by most industrial players. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Effect of simple cook and defatten processing method on the protein ...

    African Journals Online (AJOL)

    The cooked-defatted diet (CDD) and casilan diet (CAD) supported growth while Nitrogen-free diet (NFD), raw diet (RD) and cooked diet (CD) did not support growth. The protein efficiency ratio (PER), net protein utilization (NPU), apparent and true digestibility of the raw diets were very low (-4.01 ± 1.66, 48.35 ± 7.64, 44.90 ...

  2. Staged-probability strategy of processing shotgun proteomic data to discover more functionally important proteins.

    Science.gov (United States)

    Xu, Hong; Ma, Guijun; Tan, Qingqiao; Zhou, Qiang; Su, Wen; Li, Rongxiu

    2012-02-01

    Biologically important proteins related to membrane receptors, signal transduction, regulation, transcription, and translation are usually low in abundance and identified with low probability in mass spectroscopy (MS)-based analyses. Most valuable proteomics information on them were hitherto discarded due to the application of excessively strict data filtering for accurate identification. In this study, we present a staged-probability strategy for assessing proteomic data for potential functionally important protein clues. MS-based protein identifications from the second (L2) and third (L3) layers of the cascade affinity fractionation using the Trans-Proteomic Pipeline software were classified into three probability stages as 1.00-0.95, 0.95-0.50, and 0.50-0.20 according to their distinctive identification correctness rates (i.e. 100%-95%, 95%-50%, and 50%-20%, respectively). We found large data volumes and more functionally important proteins located at the previously unacceptable lower probability stages of 0.95-0.50 and 0.50-0.20 with acceptable correctness rate. More importantly, low probability proteins in L2 were verified to exist in L3. Together with some MS spectrogram examples, comparisons of protein identifications of L2 and L3 demonstrated that the staged-probability strategy could more adequately present both quantity and quality of proteomic information, especially for researches involving biomarker discovery and novel therapeutic target screening.

  3. 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.

  4. Downstream Processing Technologies/Capturing and Final Purification : Opportunities for Innovation, Change, and Improvement. A Review of Downstream Processing Developments in Protein Purification.

    Science.gov (United States)

    Singh, Nripen; Herzer, Sibylle

    2017-08-10

    Increased pressure on upstream processes to maximize productivity has been crowned with great success, although at the cost of shifting the bottleneck to purification. As drivers were economical, focus is on now on debottlenecking downstream processes as the main drivers of high manufacturing cost. Devising a holistically efficient and economical process remains a key challenge. Traditional and emerging protein purification strategies with particular emphasis on methodologies implemented for the production of recombinant proteins of biopharmaceutical importance are reviewed. The breadth of innovation is addressed, as well as the challenges the industry faces today, with an eye to remaining impartial, fair, and balanced. In addition, the scope encompasses both chromatographic and non-chromatographic separations directed at the purification of proteins, with a strong emphasis on antibodies. Complete solutions such as integrated USP/DSP strategies (i.e., continuous processing) are discussed as well as gains in data quantity and quality arising from automation and high-throughput screening (HTS). Best practices and advantages through design of experiments (DOE) to access a complex design space such as multi-modal chromatography are reviewed with an outlook on potential future trends. A discussion of single-use technology, its impact and opportunities for further growth, and the exciting developments in modeling and simulation of DSP rounds out the overview. Lastly, emerging trends such as 3D printing and nanotechnology are covered. Graphical Abstract Workflow of high-throughput screening, design of experiments, and high-throughput analytics to understand design space and design space boundaries quickly. (Reproduced with permission from Gregory Barker, Process Development, Bristol-Myers Squibb).

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

    International Nuclear Information System (INIS)

    Isidro, Anabela; Henriques, Adriano O.; Tavares, Paulo

    2004-01-01

    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

  6. Pumpkin (Cucurbita maxima) seed proteins: sequential extraction processing and fraction characterization.

    Science.gov (United States)

    Rezig, Leila; Chibani, Farhat; Chouaibi, Moncef; Dalgalarrondo, Michèle; Hessini, Kamel; Guéguen, Jacques; Hamdi, Salem

    2013-08-14

    Seed proteins extracted from Tunisian pumpkin seeds ( Cucurbita maxima ) were investigated for their solubility properties and sequentially extracted according to the Osborne procedure. The solubility of pumpkin proteins from seed flour was greatly influenced by pH changes and ionic strength, with higher values in the alkaline pH regions. It also depends on the seed defatting solvent. Protein solubility was decreased by using chloroform/methanol (CM) for lipid extraction instead of pentane (P). On the basis of differential solubility fractionation and depending on the defatting method, the alkali extract (AE) was the major fraction (42.1 (P), 22.3% (CM)) compared to the salt extract (8.6 (P), 7.5% (CM)). In salt, alkali, and isopropanol extracts, all essential amino acids with the exceptions of threonine and lysine met the minimum requirements for preschool children (FAO/WHO/UNU). The denaturation temperatures were 96.6 and 93.4 °C for salt and alkali extracts, respectively. Pumpkin protein extracts with unique protein profiles and higher denaturation temperatures could impart novel characteristics when used as food ingredients.

  7. Synthesis and processing of ovine trophoblast protein-1 and bovine trophoblast protein-1, conceptus secretory proteins involved in the maternal recognition of pregnancy.

    Science.gov (United States)

    Anthony, R V; Helmer, S D; Sharif, S F; Roberts, R M; Hansen, P J; Thatcher, W W; Bazer, F W

    1988-09-01

    Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) are newly discovered proteins produced by embryonic tissues for a limited period in early gestation. They appear to act as agents that prevent regression of the corpus luteum during early pregnancy in the ewe and cow. Ovine TP-1 [mol wt (Mr), 17,000] consists of three or four isoelectric variants (pI 5.4-5.7), whereas bTP-1, which cross-reacts with antiserum to oTP-1, is found as two predominant Mr classes (Mr, 22,000 and 24,000), each with several isoelectric variants (in the pI range 6.3-6.8). Cell-free translation of ovine conceptus mRNA yields pre-oTP-1 also consists of three or four isoelectric variants, assumed to have arisen by translation of multiple mRNA species. Ovine TP-1 is not glycosylated. When bovine conceptus mRNA is translated, a group of four or five isoforms of pre-bTP-1 are generated, each with a Mr of 19,000. In the presence of microsomes the Mr shifts upward to about 21,500. Bovine conceptuses cultured in presence of either [3H]glucosamine or [3H]mannose incorporate label into both size classes of bTP-1 (Mr, 22,000 and 24,000). Culture in presence of [35S]methionine and tunicamycin gave rise to a nonglycosylated form of bTP-1 with an apparent Mr of 18,000. Treatment of [35S]methionine-labeled bTP-1 with either endoglycosidase-H or peptide:N-glycosidase F yielded products with Mr of 17,000 and 16,000, respectively. bTP-1, although functionally and structurally related to oTP-1, appears to be a glycoprotein carrying at least two Asn-linked oligosaccharides. The two Mr classes of bTP-1 arise as a result of differences in either the number or structure of the carbohydrate chains. Like oTP-1, bTP-1 is probably translated from multiple mRNA species.

  8. Loss of protein kinase 2 subunit alpha 2 (CK2α') effect ram sperm function after freezing and thawing process.

    Science.gov (United States)

    He, Yuxuan; Li, Hongyan; Wang, Ke; Zhang, Yong; Zhao, Xingxu

    2017-06-01

    Protein kinase 2 subunit alpha 2 (CK2α'), a serine/threonine-selective protein kinase, is associated with sperm apoptosis. However, the presence of CK2α' in ram sperm during the freezing-thawing process has not been previously reported. Thus, this study was conducted to determine the effect of cryopreservation on the association between CK2α' and ram sperm function. Sperm variables, including sperm motility, DNA damage and acrosome integrity, were measured in fresh (F), cooled (CO) and freeze-thawed (FT) sperm. Sperm proteins and total mRNA were extracted form cells of each group, and subjected to western blot and real-time PCR analysis for detection of CK2α' proteins and relative abundance of mRNA. The distribution pattern of CK2α' protein in ram sperm was also monitored in each group using an immunofluorescence technique. The results provided evidence that the freeze-thaw process has an impact on ram sperm variables, and the normalized CK2α' protein and relative abundance of CK2α' mRNA were both significantly less in FT than F sperm. The amount of CK2α' in FT extended seminal plasma was increased as compared with F samples. Furthermore, immunofluorescence revealed that CK2α' was distributed throughout the acrosome of ram sperm. The association of CK2α' with DNA damage and acrosome integrity was confirmed using Pearson's linear correlation. In conclusion, the understanding the molecular effects of cryopreservation on CK2α' in ram sperm could provide insight into methods for improving fertility associated with frozen-thawed ram sperm. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. 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.

    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...... 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....

  10. The foodomics approach for the evaluation of protein bioaccessibility in processed meat upon in vitro digestion.

    Science.gov (United States)

    Bordoni, Alessandra; Laghi, Luca; Babini, Elena; Di Nunzio, Mattia; Picone, Gianfranco; Ciampa, Alessandra; Valli, Veronica; Danesi, Francesca; Capozzi, Francesco

    2014-06-01

    The present work describes a foodomics protocol coupling an in vitro static simulation of digestion to a combination of omics techniques, to grant an overview of the protein digestibility of a meat-based food, namely Bresaola. The proteolytic activity mediated by the digestive enzymes is evaluated through Bradford and SDS-PAGE assays, combined to NMR relaxometry and spectroscopy, to obtain information ranging from the microscopic to the molecular level, respectively. The simple proteomics tool adopted here points out that a clear increase of bioaccessible proteins occurs in the gastric phase, rapidly disappearing during the following duodenal digestion. However, SDS-PAGE and the Bradford assay cannot follow the fate of the digested proteins when the products are sized meat matrix. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    International Nuclear Information System (INIS)

    Delamere, N.A.; Socci, R.R.; King, K.L.

    1990-01-01

    The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin

  12. Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein

    OpenAIRE

    Botto, Laura; Cunati, Diana; Coco, Silvia; Sesana, Silvia; Bulbarelli, Alessandra; Biasini, Emiliano; Colombo, Laura; Negro, Alessandro; Chiesa, Roberto; Masserini, Massimo; Palestini, Paola

    2014-01-01

    The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC bet...

  13. LPA19, a Psb27 Homolog in Arabidopsis thaliana, Facilitates D1 Protein Precursor Processing during PSII Biogenesis*

    Science.gov (United States)

    Wei, Lili; Guo, Jinkui; Ouyang, Min; Sun, Xuwu; Ma, Jinfang; Chi, Wei; Lu, Congming; Zhang, Lixin

    2010-01-01

    The biogenesis and assembly of photosystem II (PSII) are mainly regulated by the nuclear-encoded factors. To further identify the novel components involved in PSII biogenesis, we isolated and characterized a high chlorophyll fluorescence low psii accumulation19 (lpa19) mutant, which is defective in PSII biogenesis. LPA19 encodes a Psb27 homolog (At1g05385). Interestingly, another Psb27 homolog (At1g03600) in Arabidopsis was revealed to be required for the efficient repair of photodamaged PSII. These results suggest that the Psb27 homologs play distinct functions in PSII biogenesis and repair in Arabidopsis. Chloroplast protein labeling assays showed that the C-terminal processing of D1 in the lpa19 mutant was impaired. Protein overlay assays provided evidence that LPA19 interacts with D1, and coimmunoprecipitation analysis demonstrated that LPA19 interacts with mature D1 (mD1) and precursor D1 (pD1). Moreover, LPA19 protein was shown to specifically interact with the soluble C terminus present in the precursor and mature D1 through yeast two-hybrid analyses. Thus, these studies suggest that LPA19 is involved in facilitating the D1 precursor protein processing in Arabidopsis. PMID:20444695

  14. TRIM39 is a MOAP-1-binding protein that stabilizes MOAP-1 through inhibition of its poly-ubiquitination process.

    Science.gov (United States)

    Lee, San San; Fu, Nai Yang; Sukumaran, Sunil K; Wan, Kah Fei; Wan, Qian; Yu, Victor C

    2009-04-15

    Bax, a multi-domain pro-apoptotic Bcl-2 family member, is a key regulator for the release of apoptogenic factors from mitochondria. MOAP-1, which was first isolated from a screen for Bax-associating proteins, interacts with Bax upon apoptotic induction. MOAP-1 is a short-lived protein that is constitutively degraded by the ubiquitin-proteasome system. Apoptotic stimuli upregulate MOAP-1 rapidly through inhibition of its poly-ubiquitination process. However, cellular factors that regulate the stability of MOAP-1 have not yet been identified. In this study, we report the identification of TRIM39 as a MOAP-1-binding protein. TRIM39 belongs to a family of proteins characterized by a Tripartite Motif (TRIM), consisting of RING domain, B-box and coiled-coil domain. Several TRIM family members are known to demonstrate E3 ubiquitin ligase activity. Surprisingly, TRIM39 significantly extends the half-life of MOAP-1 by inhibiting its poly-ubiquitination process. In agreement with its effect on enhancing MOAP-1 stability, TRIM39 sensitizes cells to etoposide-induced apoptosis. Conversely, knockdown of TRIM39 reduces the sensitivity of cells to etoposide-stimulated apoptosis. Furthermore, TRIM39 elevates the level of MOAP-1 in mitochondria and promotes cytochrome c release from isolated mitochondria stimulated by recombinant Bax. Together, these data suggest that TRIM39 can promote apoptosis signalling through stabilization of MOAP-1.

  15. Enrichment and Purification of Casein Glycomacropeptide from Whey Protein Isolate Using Supercritical Carbon Dioxide Processing and Membrane Ultrafiltration

    Directory of Open Access Journals (Sweden)

    Laetitia M. Bonnaillie

    2014-01-01

    Full Text Available Whey protein concentrates (WPC and isolates (WPI, comprised mainly of β-lactoglobulin (β-LG, α-lactalbumin (α-LA and casein glycomacropeptide (GMP, are added to foods to boost nutritional and functional properties. Supercritical carbon dioxide (SCO2 has been shown to effectively fractionate WPC and WPI to obtain enriched fractions of α-LA and β-LG, thus creating new whey ingredients that exploit the properties of the individual component proteins. In this study, we used SCO2 to further fractionate WPI via acid precipitation of α-LA, β-LG and the minor whey proteins to obtain GMP-enriched solutions. The process was optimized and α-LA precipitation maximized at low pH and a temperature (T ≥65 °C, where β-LG with 84% purity and GMP with 58% purity were obtained, after ultrafiltration and diafiltration to separate β-LG from the GMP solution. At 70 °C, β-LG also precipitated with α-LA, leaving a GMP-rich solution with up to 94% purity after ultrafiltration. The different protein fractions produced with the SCO2 process will permit the design of new foods and beverages to target specific nutritional needs.

  16. Effect of processing on the in vitro and in vivo protein quality of red and green lentils (Lens culinaris).

    Science.gov (United States)

    Nosworthy, Matthew G; Medina, Gerardo; Franczyk, Adam J; Neufeld, Jason; Appah, Paulyn; Utioh, Alphonsus; Frohlich, Peter; House, James D

    2018-02-01

    In order to determine the effect of extrusion, baking and cooking on the protein quality of red and green lentils, a rodent bioassay was conducted and compared to an in vitro method of protein quality determination. On average, the Protein Digestibility-Corrected Amino Acid Score of red lentils (55.0) was higher than that of green lentils (50.8). Extruded lentil flour had higher scores (63.01 red, 57.09 green) than either cooked (57.40 red, 52.92 green) or baked (53.84 red, 47.14 green) flours. The average Digestible Indispensable Amino Acid Score of red lentils (0.54) was higher than green lentils (0.49). The Protein Efficiency Ratio of the extruded lentil flours (1.30 red, 1.34 green) was higher than that of the baked flour (0.98 red, 1.09 green). A correlation was found between in vivo and in vitro methods of determining protein digestibility (R 2 =0.8934). This work could influence selection of processing method during product development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Influence of storage and heating on protein glycation levels of processed lactose-free and regular bovine milk products.

    Science.gov (United States)

    Milkovska-Stamenova, Sanja; Hoffmann, Ralf

    2017-04-15

    Thermal treatment preserves the microbiological safety of milk, but also induces Maillard reactions modifying for example proteins. The purpose of this study was evaluating the influence of consumer behaviors (storage and heating) on protein glycation degrees in bovine milk products. Lactosylation and hexosylation sites were identified in ultra-high temperature (UHT), lactose-free pasteurized, and lactose-free UHT milk (ULF) and infant formula (IF) using tandem mass spectrometry (electron transfer dissociation). Overall, 303 lactosylated and 199 hexosylated peptides were identified corresponding to 170 lactosylation (31 proteins) and 117 hexosylation sites (25 proteins). In quantitative terms, storage increased lactosylation up to fourfold in UHT and IF and hexosylation up to elevenfold in ULF and threefold in IF. These levels increased additionally twofold when the stored samples were heated (40°C). In conclusion, storage and heating appear to influence protein glycation levels in milk at similar or even higher degrees than industrial processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  19. Detection of soy proteins in processed foods: Literature overview and new experimental work

    NARCIS (Netherlands)

    Koppelman, S.J.; Lakemond, C.M.M.; Vlooswijk, R.; Hefle, S.L.

    2004-01-01

    Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory,

  20. Food protein aggregates as vitamin-matrix carriers: impact of processing conditions.

    Science.gov (United States)

    Relkin, Perla; Shukat, Rizwan

    2012-10-15

    We studied the ability of protein aggregates for loading and protection of α-tocopherol, a model of heat- and light-sensitive bioactive compounds. Aqueous dispersions of whey proteins (4.5 wt.%, pH 6.7) in the absence and presence of α-tocopherol (4 wt.%) were prepared using an ultradisperser (10,000 rpm for 10 min and 65 °C), and then submitted to further high-pressure homogenisation (HPH) at 300 or 1200 bar for 12 cycles. Relative to free-vitamin dispersions, increasing HPH conditions in the presence of vitamin led to higher protein denaturation, more tryptophan quenching and wavelength blue-shift (by 10nm), in parallel with increased zeta potential values (by -10 mV), particle sizes (by 50%), and newly formed protein dimers, trimers and high molecular weight aggregates. As a result, the degree of vitamin degradation under increasing HPH and long-term storage was shown to decrease from 66% (ultradisper) to 50%, or to 30% (subject to further treatments at 300 or 1200 bar, respectively). Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Process for the production of protein enriched fractions from vegetable materials

    NARCIS (Netherlands)

    Dijkink, B.H.; Willemsen, J.H.A.

    2006-01-01

    The present invention provides a method for the production of a protein enriched fraction and a fibre enriched fraction from a vegetable material, wherein the vegetable material comprises a total fat content of 0.1 to 22.0 % by dry weight of the total vegetable material and a total starch content of

  2. Network-Based Methods for Identifying Key Active Proteins in the Extracellular Electron Transfer Process in Shewanella oneidensis MR-1

    OpenAIRE

    Ding, Dewu; Sun, Xiao

    2018-01-01

    Shewanella oneidensis MR-1 can transfer electrons from the intracellular environment to the extracellular space of the cells to reduce the extracellular insoluble electron acceptors (Extracellular Electron Transfer, EET). Benefiting from this EET capability, Shewanella has been widely used in different areas, such as energy production, wastewater treatment, and bioremediation. Genome-wide proteomics data was used to determine the active proteins involved in activating the EET process. We iden...

  3. In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography.

    Science.gov (United States)

    Großhans, Steffen; Rüdt, Matthias; Sanden, Adrian; Brestrich, Nina; Morgenstern, Josefine; Heissler, Stefan; Hubbuch, Jürgen

    2018-03-05

    Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q 2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q 2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Nanocomposited coatings produced by laser-assisted process to prevent silicone hydogels from protein fouling and bacterial contamination

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Guobang; Chen, Yi; Zhang, Jin, E-mail: jzhang@eng.uwo.ca

    2016-01-01

    Graphical abstract: Nanocomposited-coating was deposited on silicone hydrogel by using the matrix-assisted pulsed laser evaporation (MAPLE) process. The ZnO–PEG nanocomposited coating reduces over 50% protein absorption on silicone hydrogel, and can inhibit the bacterial growth efficiently. - Highlights: • We developed a nanocomposited coating to prevent silicone hydrogel from biofouling. • Matrix-assisted pulsed laser evaporation can deposit inorganic–organic nanomaterials. • The designed nanocomposited coating reduces protein absorption by over 50%. • The designed nanocomposited coating shows significant antimicrobial efficiency. - Abstract: Zinc oxide (ZnO) nanoparticles incorporating with polyethylene glycol (PEG) were deposited together on the surface of silicone hydrogel through matrix-assisted pulsed laser evaporation (MAPLE). In this process, frozen nanocomposites (ZnO–PEG) in isopropanol were irradiated under a pulsed Nd:YAG laser at 532 nm for 1 h. Our results indicate that the MAPLE process is able to maintain the chemical backbone of polymer and prevent the nanocomposite coating from contamination. The ZnO–PEG nanocomposited coating reduces over 50% protein absorption on silicone hydrogel. The cytotoxicity study shows that the ZnO–PEG nanocomposites deposited on silicone hydrogels do not impose the toxic effect on mouse NIH/3T3 cells. In addition, MAPLE-deposited ZnO–PEG nanocomposites can inhibit the bacterial growth significantly.

  5. Comparing and combining implicit ligand sampling with multiple steered molecular dynamics to study ligand migration processes in heme proteins.

    Science.gov (United States)

    Forti, Flavio; Boechi, Leonardo; Estrin, Dario A; Marti, Marcelo A

    2011-07-30

    The ubiquitous heme proteins perform a wide variety of tasks that rely on the subtle regulation of their affinity for small ligands like O2, CO, and NO. Ligand affinity is characterized by kinetic association and dissociation rate constants, that partially depend on ligand migration between the solvent and active site, mediated by the presence of internal cavities or tunnels. Different computational methods have been developed to study these processes which can be roughly divided in two strategies: those costly methods in which the ligand is treated explicitly during the simulations, and the free energy landscape of the process is computed; and those faster methods that use prior computed Molecular Dynamics simulation without the ligand, and incorporate it afterwards, called implicit ligand sampling (ILS) methods. To compare both approaches performance and to provide a combined protocol to study ligand migration in heme proteins, we performed ILS and multiple steered molecular dynamics (MSMD) free energy calculations of the ligand migration process in three representative and well theoretically and experimentally studied cases that cover a wide range of complex situations presenting a challenging benchmark for the aim of the present work. Our results show that ILS provides a good description of the tunnel topology and a reasonable approximation to the free energy landscape, while MSMD provides more accurate and detailed free energy profile description of each tunnel. Based on these results, a combined strategy is presented for the study of internal ligand migration in heme proteins. Copyright © 2011 Wiley Periodicals, Inc.

  6. Use of vaccinia virus vectors to study protein processing in human disease. Normal nerve growth factor processing and secretion in cultured fibroblasts from patients with familial dysautonomia.

    Science.gov (United States)

    Edwards, R H; Rutter, W J

    1988-07-01

    Familial dysautonomia is a hereditary disorder that affects autonomic and sensory neurons. Nerve growth factor (NGF) is required for the normal development of sympathetic and sensory neurons and it has been postulated that an abnormality involving NGF may be responsible for familial dysautonomia. Previous studies have shown that the beta-NGF gene is not linked to the disease. However, NGF appears to be abnormal by immunochemical assays; the putative altered form of NGF could result from a disturbance in the processing pathway. To study the processing of the 35-kD glycosylated NGF precursor and the secretion of NGF in familial dysautonomia, we have employed a recombinant vaccinia virus vector to express high levels of NGF mRNA in primary fibroblast cultures from patients with the disorder; the processing pathway was then studied directly. Cells from several unrelated patients all produce the same 35-kD NGF precursor, process this normally to NGF within the cell, and release NGF into the medium. There are no differences in the ability of cells from patients and from unaffected relatives to process and secrete NGF. The use of similar recombinant vaccinia virus vectors to express proteins at high level in primary cell lines should facilitate the detection of posttranslational processing defects in a variety of human disorders.

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

    Science.gov (United States)

    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 degradation of silk film by protease. Model compounds spanning a range of physical-chemical properties generally showed an inverse relationship between degumming time and release rate through a varied degumming time silk coating. Degumming provides a useful control point to manipulate silk’s material properties. PMID:23349062

  8. New learning while consolidating memory during sleep is actively blocked by a protein synthesis dependent process.

    Science.gov (United States)

    Levy, Roi; Levitan, David; Susswein, Abraham J

    2016-12-06

    Brief experiences while a memory is consolidated may capture the consolidation, perhaps producing a maladaptive memory, or may interrupt the consolidation. Since consolidation occurs during sleep, even fleeting experiences when animals are awakened may produce maladaptive long-term memory, or may interrupt consolidation. In a learning paradigm affecting Aplysia feeding, when animals were trained after being awakened from sleep, interactions between new experiences and consolidation were prevented by blocking long-term memory arising from the new experiences. Inhibiting protein synthesis eliminated the block and allowed even a brief, generally ineffective training to produce long-term memory. Memory formation depended on consolidative proteins already expressed before training. After effective training, long term memory required subsequent transcription and translation. Memory formation during the sleep phase was correlated with increased CREB1 transcription, but not CREB2 transcription. Increased C/EBP transcription was a correlate of both effective and ineffective training and of treatments not producing memory.

  9. Structural changes on a molecular basis of canola meal by conditioning temperature and time during pelleting process in relation to physiochemical (energy and protein) properties relevant to ruminants.

    Science.gov (United States)

    Huang, Xuewei; Zhang, Huihua; Yu, Peiqiang

    2017-01-01

    The objectives of this study were: (1) To investigate the effects of conditioning temperature (70, 80, 90°C), time (30, 60 sec), and interaction (temperature × time) during the pelleting process on internal protein molecular structure changes of the co-products; (2) To identify differences in protein molecular structures among pellets that were processed under different conditions, and between unprocessed mash and pellets; 3) To quantify protein molecular structure changes in relation to predicted energy and protein utilization in dairy cows. The final goal of this program was to show how processing conditions changed internal feed structure on a molecular basis and how molecular structure changes induced by feed processing affected feed milk value in dairy cows. The hypothesis in this study was that processing-induced protein inherent structure changes affected energy and protein availability in dairy cattle and the sensitivity and response of protein internal structure to the different pelleting process conditions could be detected by advanced molecular spectroscopy. The protein molecular structures, amides I and II, amide I to II ratios, α-helix structure, β-sheet structure, and α to β structure ratios, were determined using the advanced vibrational molecular spectroscopy (ATR-FT/IR). The energy values were determined using NRC2001 summary approach in terms of total digestible nutrients, metabolizable and net energy for lactation. The protein and carbohydrate subfactions that are related to rumen degradation characteristics and rumen undegraded protein supply were determined using updated CNCPS system. The experiment design was a RCBD and the treatment design was a 3x2 factorial design. The results showed that pelleting induced changes in protein molecular structure. The sensitivity and response of protein inherent structure to the pelleting depended on the conditioning temperature and time. The protein molecular structure changes were correlated (P < 0

  10. The influence of different salting processes on protein loss of cuttlefish (Sepia officinalis.

    Directory of Open Access Journals (Sweden)

    Mustafa Ünlüsayın

    2015-12-01

    Full Text Available Sübye (Sepia officinalis’nin protein kayıpları üzerine farklı tuzlama yöntemlerinin etkileri. Bu çalışmanın amacı; farklı tuzlama metotlarıyla tuzlanan sübye (Sepia officinalis L., 1758’nin protein kayıplarını araştırmaktır. %8 ve %20 konsantrasyonlarında NaCl içeren iki farklı tuz solüsyonu çalışılmıştır. Üçüncü yöntem olan kuru tuzlama yöntemi ise balık yüzeyinin tamamı tuz ile kaplanarak yapılmıştır. Taze ve %8’lik solüsyonla tuzlanan sübyelerin nem içeriğindeki değişimler önemsizdi (P>0,05. %8 ve %20’lik konsantrasyonlarla tuzlanan sübyelerin protein içeriğindeki (kuru ağırlık üzerinden değişimler önemsiz (P>0,05 olarak belirlenmiştir. Tuzlamadan sonra tüm gruplarda sübyelerin toplam protein içeriği azalmıştır (P

  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. Downstream processing of biopharmaceutical proteins produced in plants: The pros and cons of flocculants

    OpenAIRE

    Buyel, Johannes Felix; Fischer, Rainer

    2014-01-01

    All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilit...

  13. Role of lipid rafts and GM1 in the segregation and processing of prion protein.

    Directory of Open Access Journals (Sweden)

    Laura Botto

    Full Text Available The prion protein (PrPC is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical, stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.

  14. Role of lipid rafts and GM1 in the segregation and processing of prion protein.

    Science.gov (United States)

    Botto, Laura; Cunati, Diana; Coco, Silvia; Sesana, Silvia; Bulbarelli, Alessandra; Biasini, Emiliano; Colombo, Laura; Negro, Alessandro; Chiesa, Roberto; Masserini, Massimo; Palestini, Paola

    2014-01-01

    The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.

  15. Application of Quality by Design to the characterization of the cell culture process of an Fc-Fusion protein.

    Science.gov (United States)

    Rouiller, Yolande; Solacroup, Thomas; Deparis, Véronique; Barbafieri, Marco; Gleixner, Ralf; Broly, Hervé; Eon-Duval, Alex

    2012-06-01

    The production bioreactor step of an Fc-Fusion protein manufacturing cell culture process was characterized following Quality by Design principles. Using scientific knowledge derived from the literature and process knowledge gathered during development studies and manufacturing to support clinical trials, potential critical and key process parameters with a possible impact on product quality and process performance, respectively, were determined during a risk assessment exercise. The identified process parameters were evaluated using a design of experiment approach. The regression models generated from the data allowed characterizing the impact of the identified process parameters on quality attributes. The main parameters having an impact on product titer were pH and dissolved oxygen, while those having the highest impact on process- and product-related impurities and variants were pH and culture duration. The models derived from characterization studies were used to define the cell culture process design space. The design space limits were set in such a way as to ensure that the drug substance material would consistently have the desired quality. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Shuai Wang

    2016-05-01

    Full Text Available Plants have varying abilities to tolerate chilling (low but not freezing temperatures, and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.

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

    International Nuclear Information System (INIS)

    Gutfilen, Bianca

    1989-01-01

    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)

  18. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity.

    Science.gov (United States)

    Tustian, Andrew D; Endicott, Christine; Adams, Benjamin; Mattila, John; Bak, Hanne

    2016-01-01

    There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.

  19. Influence of different processing treatments on the detectability of nucleic acid and protein targets in transgenic soybean meal.

    Science.gov (United States)

    Tian, Fang; Guan, Qingfeng; Wang, Xiumin; Teng, Da; Wang, Jianhua

    2014-04-01

    Influences of dry heating, wet heating, and extrusion on the degradation of DNA and protein from transgenic soybean meal (TSM) were analyzed using qualitative PCR, quantitative real-time PCR (qPCR), indirect enzyme-linked immunosorbent assay (iELISA), and Western blot. The 414-bp Lectin gene was thoroughly degraded after dry heating between 75 and 90 °C for 30 min, wet heating, and extrusion at 165 °C with 39 % moisture content. The 483-bp CP4-EPSPS gene was not detected after dry heating, wet heating, and extrusion at 120 °C with 39 % moisture content. The degradation ratios of both Lectin and CP4-EPSPS genes increased from 0.4 to 92.1 % and 6.1 to 84.0 % as temperatures rose from 90 to 165 °C. iELISA results of the extruded TSM showed that the CP4-EPSPS protein content was reduced from 4.19 to 0.54 % as temperatures rose from 90 to 150 °C and was not detectable at 165 °C. Western blot results also showed the degradation of CP4-CPSPS protein after extrusion. Our results showed that temperature played an essential role in DNA and protein degradation, and the content of genetically modified organism (GMO) products may be changed after processing and could not reflect the initial content of the products.

  20. 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.

  1. Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells.

    Science.gov (United States)

    Rathore, Anurag Singh; Sobacke, S E; Kocot, T J; Morgan, D R; Dufield, R L; Mozier, N M

    2003-08-21

    Analyses of crude samples from biotechnology processes are often required in order to demonstrate that residual host cell impurities are reduced or eliminated during purification. In later stages of development, as the processes are further developed and finalized, there is a tremendous volume of testing required to confirm the absence of residual host cell proteins (HCP) and DNA. Analytical tests for these components are very challenging since (1). they may be present at levels that span a million-fold range, requiring substantial dilutions; (2). are not a single component, often existing as fragments and a variety of structures; (3). require high sensitivity for final steps in process; and (4). are present in very complex matrices including other impurities, the product, buffers, salts and solvents. Due to the complex matrices and the variety of potential analytes, the methods of analysis are not truly quantitative for all species. Although these limitations are well known, the assays are still very much in demand since they are required for approval of new products. Methods for final products, described elsewhere, focus on approaches to achieve regulatory requirements. The study described herein will describe the technical rationale for measuring the clearance of HCP and DNA in the entire bioprocessing to purification from an Escherichia coli-derived expression system. Three analytical assays, namely, reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA), and Threshold Total DNA Assay, were utilized to quantify the protein product, HCP and DNA, respectively. Product quantification is often required for yield estimation and is useful since DNA and HCP results are best expressed as a ratio to product for calculation of relative purification factors. The recombinant E. coli were grown to express the protein of interest as insoluble inclusion bodies (IB) within the cells. The IB were isolated by repeated

  2. The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.

    Directory of Open Access Journals (Sweden)

    Luis G Morello

    Full Text Available 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.

  3. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Science.gov (United States)

    Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

    2017-01-01

    There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

  4. Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

    Science.gov (United States)

    Ader-Ebert, Nadine; Khosravi, Mojtaba; Herren, Michael; Avila, Mislay; Alves, Lisa; Bringolf, Fanny; Örvell, Claes; Langedijk, Johannes P.; Zurbriggen, Andreas; Plemper, Richard K.; Plattet, Philippe

    2015-01-01

    Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the

  5. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum

    OpenAIRE

    Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A.; Salic, Adrian

    2011-01-01

    The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is s...

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

    Energy Technology Data Exchange (ETDEWEB)

    O, Shepelenko S; S, Salnikov A; V, Rak S; P, Goncharova E; B, Ryzhikov A, E-mail: shep@vector.nsc.r, E-mail: shep@ngs.r [Federal State Research Institution State Research Center of Virology and Biotechnology VECTOR of the Federal Service for Surveillance in Consumer Rights Protection and Human Well-being (FSRI SRC VB VECTOR) Koltsovo, Novosibirsk Region (Russian Federation)

    2009-06-01

    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

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

    International Nuclear Information System (INIS)

    O, Shepelenko S; S, Salnikov A; V, Rak S; P, Goncharova E; B, Ryzhikov A

    2009-01-01

    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

  8. Calretinin: from a simple Ca2+ buffer to a multifunctional protein implicated in many biological processes

    Directory of Open Access Journals (Sweden)

    Beat eSchwaller

    2014-02-01

    Full Text Available The hexa-EF-hand Ca2+-binding protein calretinin (CR is predominantly expressed in specific neurons of the central and peripheral nervous system. However, CR expression is also observed in non-neuronal cells, e.g. during embryonic development and in mesothelioma cells. Of the 6 EF-hand domains, 5 are functional; the first 4 domains form 2 pairs showing high cooperativity within a pair that results in non-linear modulation of intracellular Ca2+ signals by CR. EF-hand domain 5 has a low affinity and represents the identified interaction site with CR-binding partners present in mouse cerebellar granule cells. CR binding to other targets including the pore-forming α1 subunit of the Ca2+ channel CaV2.1, as well as to huntingtin indicates additional Ca2+ sensor functions besides the well-known Ca2+-buffering functions. The absence of CR in cerebellar granule cells of CR-/- mice results in increased excitability and altered firing of Purkinje cells and promotes cerebellar 160-Hz oscillations impairing motor coordination. The putative role of CR in neuroprotection is still highly discussed. Altogether, CR emerges as a multi-functional protein also associated with development, i.e. cell proliferation, differentiation and cell death.

  9. Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements.

    Science.gov (United States)

    Zieger, Martin; Mayer, Andreas

    2012-09-01

    Yeast vacuoles fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. Here we analyze osmotically induced vacuole fragmentation by time-lapse microscopy. Small fragmentation products originate directly from the large central vacuole. This happens by asymmetrical scission rather than by consecutive equal divisions. Fragmentation occurs in two distinct phases. Initially, vacuoles shrink and generate deep invaginations that leave behind tubular structures in their vicinity. Already this invagination requires the dynamin-like GTPase Vps1p and the vacuolar proton gradient. Invaginations are stabilized by phosphatidylinositol 3-phosphate (PI(3)P) produced by the phosphoinositide 3-kinase complex II. Subsequently, vesicles pinch off from the tips of the tubular structures in a polarized manner, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol-3,5-bisphosphate and the Fab1 complex. It is accelerated by the PI(3)P- and phosphatidylinositol 3,5-bisphosphate-binding protein Atg18p. Thus vacuoles fragment in two steps with distinct protein and lipid requirements.

  10. First Experimental Assessment of Protein Intrinsic Disorder Involvement in an RNA Virus Natural Adaptive Process.

    Science.gov (United States)

    Charon, Justine; Barra, Amandine; Walter, Jocelyne; Millot, Pauline; Hébrard, Eugénie; Moury, Benoît; Michon, Thierry

    2018-01-01

    Intrinsic disorder (ID) in proteins is defined as a lack of stable structure in physiological conditions. Intrinsically disordered regions (IDRs) are highly abundant in some RNA virus proteomes. Low topological constraints exerted on IDRs are expected to buffer the effect of numerous deleterious mutations and could be related to the remarkable adaptive potential of RNA viruses to overcome resistance of their host. To experimentally test this hypothesis in a natural pathosystem, a set of four variants of Potato virus Y (PVY; Potyvirus genus) containing various ID degrees in the Viral genome-linked (VPg) protein, a key determinant of potyvirus adaptation, was designed. To estimate the ID contribution to the VPg-based PVY adaptation, the adaptive ability of the four PVY variants was monitored in the pepper host (Capsicum annuum) carrying a recessive resistance gene. Intriguingly, the two mutants with the highest ID content displayed a significantly higher ability to restore infection in the resistant host, whereas the less intrinsically disordered mutant was unable to restore infection. The role of ID on virus adaptation may be due either to a larger exploration of evolutionary pathways or the minimization of fitness penalty caused by resistance-breaking mutations. This pioneering study strongly suggests the positive impact of ID in an RNA virus adaptive capacity. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Elg1, the major subunit of an alternative RFC complex, interacts with SUMO-processing proteins.

    Science.gov (United States)

    Parnas, Oren; Amishay, Rona; Liefshitz, Batia; Zipin-Roitman, Adi; Kupiec, Martin

    2011-09-01

    PCNA is a homotrimeric ring with important roles in DNA replication and repair. PCNA is loaded and unloaded by the RFC complex, which is composed of five subunits (Rfc1-5). Three additional complexes that share with RFC the small subunits (Rfc2-5) and contain alternative large subunits were found in yeast and other eukaryotes. We have recently reported that one of these, the Elg1-RFC complex, interacts with SUMOylated PCNA and may play a role in its unloading during DNA repair. Here we report that a yeast-two-hybrid screen with the N terminus of Elg1(which interacts with SUMOylated PCNA) uncovered interactions with proteins that belong to the SUMO pathway, including Slx5 and Slx8, which form an E3 ubiquitin ligase that ubiquitinates SUMOylated proteins. Mutations in SLX5 result in a genomic instability phenotype similar to that of elg1 mutants. The physical interaction between the N terminus of Elg1 and Slx5 is mediated by poly-SUMO chains but not by PCNA modifications, and requires Siz2, but not Siz1, activity. Thus our results highlight the many important roles played by Elg1, some of which are PCNA-dependent and some PCNA-independent. © 2011 Landes Bioscience

  12. New learning while consolidating memory during sleep is actively blocked by a protein synthesis dependent process

    Science.gov (United States)

    Levy, Roi; Levitan, David; Susswein, Abraham J

    2016-01-01

    Brief experiences while a memory is consolidated may capture the consolidation, perhaps producing a maladaptive memory, or may interrupt the consolidation. Since consolidation occurs during sleep, even fleeting experiences when animals are awakened may produce maladaptive long-term memory, or may interrupt consolidation. In a learning paradigm affecting Aplysia feeding, when animals were trained after being awakened from sleep, interactions between new experiences and consolidation were prevented by blocking long-term memory arising from the new experiences. Inhibiting protein synthesis eliminated the block and allowed even a brief, generally ineffective training to produce long-term memory. Memory formation depended on consolidative proteins already expressed before training. After effective training, long term memory required subsequent transcription and translation. Memory formation during the sleep phase was correlated with increased CREB1 transcription, but not CREB2 transcription. Increased C/EBP transcription was a correlate of both effective and ineffective training and of treatments not producing memory. DOI: http://dx.doi.org/10.7554/eLife.17769.001 PMID:27919318

  13. 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. © 2015 Wiley Periodicals, Inc.

  14. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    Directory of Open Access Journals (Sweden)

    Elizabeth Muir

    Full Text Available There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed

  15. Probing Adsorption / Desorption Processes at the Liquid / Solid Interface: Thiols and Proteins

    Science.gov (United States)

    Campbell, Charles; Jung, Linda S.; Shumaker-Parry, Jennifer; Nelsen, K. E.; Stayton, P. S.; Gelb, M. H.; Aebersold, R.

    2001-03-01

    The adsorption of molecules from liquid solutions onto solid surfaces can be monitored with high sensitivity and fast time response by following changes in the angle or wavelength at which the surface plasmon resonance (SPR) of a thin metal film is optically excited. Simple methods convert these measured changes into adsorbate concentrations. We report here the adsorption and desorption kinetics and equilibrium coverages of a variety of species on well-characterized surfaces as determined by SPR techniques. When the diffusion constant of the adsorbing species is known in the liquid phase, the intrinsic rate constants can be determined from the kinetic results. The sticking probability, defined as the rate of adsorption per molecular collision with the surface, directly expresses the difficulty encountered by a molecule in scaling the barrier to adsorption. Its prior use has been restricted to adsorption of gases. A method extending this concept to adsorption from liquid solutions is applied to transient measurements of alkylthiol adsorption onto gold from ethanol solutions. The initial sticking probability increases from 10-8 to 10-6 with alkyl chain length, implying a stabilization of the transition state by 0.65 kJ/mol per CH_2. Since their sticking probabilities in gas phase are 1.0, the solvent increases the activation free energy by 40 kJ/mol. Applications of gold-thin-film SPR sensors in quantifying biological interactions will be described also. A gold surface containing a few biotin headgroups in a self assembled alkylthiolate monolayer of mainly oligo(ethylene glycol) (OEG) headgroups selectively adsorbs the protein streptavidin with a structure that depends on the biotin / OEG ratio. The free biotin sites in the resulting streptavidin monolayer have been used as strong linker sites for further attachment of intact, biotinylated lipid vesicles and biotinylated, double-stranded oligonucleotides to the surface. These complex biological films then provide a

  16. Entropic formulation for the protein folding process: Hydrophobic stability correlates with folding rates

    Science.gov (United States)

    Dal Molin, J. P.; Caliri, A.

    2018-01-01

    Here we focus on the conformational search for the native structure when it is ruled by the hydrophobic effect and steric specificities coming from amino acids. Our main tool of investigation is a 3D lattice model provided by a ten-letter alphabet, the stereochemical model. This minimalist model was conceived for Monte Carlo (MC) simulations when one keeps in mind the kinetic behavior of protein-like chains in solution. We have three central goals here. The first one is to characterize the folding time (τ) by two distinct sampling methods, so we present two sets of 103 MC simulations for a fast protein-like sequence. The resulting sets of characteristic folding times, τ and τq were obtained by the application of the standard Metropolis algorithm (MA), as well as by an enhanced algorithm (Mq A). The finding for τq shows two things: (i) the chain-solvent hydrophobic interactions {hk } plus a set of inter-residues steric constraints {ci,j } are able to emulate the conformational search for the native structure. For each one of the 103MC performed simulations, the target is always found within a finite time window; (ii) the ratio τq / τ ≅ 1 / 10 suggests that the effect of local thermal fluctuations, encompassed by the Tsallis weight, provides to the chain an innate efficiency to escape from energetic and steric traps. We performed additional MC simulations with variations of our design rule to attest this first result, both algorithms the MA and the Mq A were applied to a restricted set of targets, a physical insight is provided. Our second finding was obtained by a set of 600 independent MC simulations, only performed with the Mq A applied to an extended set of 200 representative targets, our native structures. The results show how structural patterns should modulate τq, which cover four orders of magnitude; this finding is our second goal. The third, and last result, was obtained with a special kind of simulation performed with the purpose to explore a

  17. 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....

  18. Data processing in neutron protein crystallography using position-sensitive detectors

    International Nuclear Information System (INIS)

    Schoenborn, B.P.

    1982-01-01

    Neutrons provide a unique probe for localizing hydrogen atoms and for distinguishing hydrogen from deuterons. Hydrogen atoms largely determine the three-dimensional structure of proteins and are responsible for many catalytic reactions. The study of hydrogen bonding and hydrogen exchange will therefore give insight into reaction mechanisms and conformational fluctuations. In addition, neutrons provide the ability to distinguish N from C and O and to allow correct orientation of groups such as histidine and glutamine. To take advantage of these unique features of neutron crystallography, one needs accurate Fourier maps depicting atomic structure to a high precision. In this paper, techniques are described for minimizing error in the observed structure factors by optimizing data collection and analysis procedures. Special attention is given to subtraction of the high background associated with hydrogen-containing molecules, which produces a disproportionately large statistical error

  19. Selective processing and metabolism of disease-causing mutant prion proteins.

    Directory of Open Access Journals (Sweden)

    Aarthi Ashok

    2009-06-01

    Full Text Available Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C. In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc form that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C to PrP(Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc in familial prion disease.

  20. 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.

  1. The use of quantitative structure-activity relationship models to develop optimized processes for the removal of tobacco host cell proteins during biopharmaceutical production.

    Science.gov (United States)

    Buyel, J F; Woo, J A; Cramer, S M; Fischer, R

    2013-12-27

    The production of recombinant pharmaceutical proteins in plants benefits from the low cost of upstream production and the greater scalability of plants compared to fermenter-based systems. Now that manufacturing processes that comply with current good manufacturing practices have been developed, plants can compete with established platforms on equal terms. However, the costs of downstream processing remain high, in part because of the dedicated process steps required to remove plant-specific process-related impurities. We therefore investigated whether the ideal strategy for the chromatographic removal of tobacco host cell proteins can be predicted by quantitative structure-activity relationship (QSAR) modeling to reduce the process development time and overall costs. We identified more than 100 tobacco proteins by mass spectrometry and their structures were reconstructed from X-ray crystallography, nuclear magnetic resonance spectroscopy and/or homology modeling data. The resulting three-dimensional models were used to calculate protein descriptors, and significant descriptors were selected based on recently-published retention data for model proteins to develop QSAR models for protein retention on anion, cation and mixed-mode resins. The predicted protein retention profiles were compared with experimental results using crude tobacco protein extracts. Because of the generic nature of the method, it can easily be transferred to other expression systems such as mammalian cells. The quality of the models and potential improvements are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. 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...

  3. 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 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 fractions. Multivariate molecular spectral analysis with cluster (CLA) and principal component analyses (PCA) showed that there are no clear distinguished clusters and

  4. Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

    Directory of Open Access Journals (Sweden)

    Danijela Koppers-Lalic

    2008-05-01

    Full Text Available Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1, pseudorabies virus (PRV, and equine herpesvirus 1 and 4 (EHV-1 and EHV-4 are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

  5. Uterine Wound Healing: A Complex Process Mediated by Proteins and Peptides.

    Science.gov (United States)

    Lofrumento, Dario D; Di Nardo, Maria A; De Falco, Marianna; Di Lieto, Andrea

    2017-01-01

    Wound healing is the process by which a complex cascade of biochemical events is responsible of the repair the damage. In vivo, studies in humans and mice suggest that healing and post-healing heterogeneous behavior of the surgically wounded myometrium is both phenotype and genotype dependent. Uterine wound healing process involves many cells: endothelial cells, neutrophils, monocytes/macrophages, lymphocytes, fibroblasts, myometrial cells as well a stem cell population found in the myometrium, myoSP (side population of myometrial cells). Transforming growth factor beta (TGF-β) isoforms, connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNF-β) are involved in the wound healing mechanisms. The increased TGF- β1/β3 ratio reduces scarring and fibrosis. The CTGF altered expression may be a factor involved in the abnormal scars formation of low uterine segment after cesarean section and of the formation of uterine dehiscence. The lack of bFGF is involved in the reduction of collagen deposition in the wound site and thicker scabs. The altered expression of TNF-β, VEGF, and PDGF in human myometrial smooth muscle cells in case of uterine dehiscence, it is implicated in the uterine healing process. The over-and under-expressions of growth factors genes involved in uterine scarring process could represent patient's specific features, increasing the risk of cesarean scar complications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. 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

    Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon...... polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model...... 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...

  7. GPU MrBayes V3.1: MrBayes on Graphics Processing Units for Protein Sequence Data.

    Science.gov (United States)

    Pang, Shuai; Stones, Rebecca J; Ren, Ming-Ming; Liu, Xiao-Guang; Wang, Gang; Xia, Hong-ju; Wu, Hao-Yang; Liu, Yang; Xie, Qiang

    2015-09-01

    We present a modified GPU (graphics processing unit) version of MrBayes, called ta(MC)(3) (GPU MrBayes V3.1), for Bayesian phylogenetic inference on protein data sets. Our main contributions are 1) utilizing 64-bit variables, thereby enabling ta(MC)(3) to process larger data sets than MrBayes; and 2) to use Kahan summation to improve accuracy, convergence rates, and consequently runtime. Versus the current fastest software, we achieve a speedup of up to around 2.5 (and up to around 90 vs. serial MrBayes), and more on multi-GPU hardware. GPU MrBayes V3.1 is available from http://sourceforge.net/projects/mrbayes-gpu/. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Characterization of the aroma of a meatlike process flavoring from soybean-based enzyme-hydrolyzed vegetable protein.

    Science.gov (United States)

    Wu, Yi-Fang G; Cadwallader, Keith R

    2002-05-08

    Defatted soybean meal was converted into enzyme-hydrolyzed vegetable protein (E-HVP) using the proteolytic enzyme Flavorzyme. Total free amino acids increased by 40-fold after enzyme hydrolysis, with leucine being the most abundant, followed by phenylalanine, lysine, glutamine/glutamic acid, and alanine. Volatile components from a meatlike process flavoring made from E-HVP were isolated by direct solvent extraction (DSE)-high vacuum transfer (HVT), dynamic headspace sampling and static headspace sampling and analyzed by gas chromatography (GC)-mass spectrometry and GC-olfactometry. Aroma extract dilution analysis was used to establish a flavor dilution chromatogram of the DSE-HVT extract. Results of these complementary techniques indicated the importance of odorants of high (hydrogen sulfide and methanethiol), intermediate (2-methyl-3-furanthiol, 3-mercapto-2-pentanone, 2-furanmethanethiol, and 3-(methylthiol)propanal) and low volatility (maltol and Furaneol) in the overall aroma of the meatlike process flavoring.

  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 < 0.05) and BBI (r = 0.89;p < 0.05) as well as BBI monomeric (r = 0.89;p < 0.05) and polymeric forms (r = 0.95;p < 0.05) in okara and in soybean were strongly correlated. Low urease index activity indicated that okara was heated adequately to inactivate 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.

  10. Effects of oil thermally processed with vegetable protein on gastrointestinal tract content transfer.

    Science.gov (United States)

    Totani, Nagao; Araki, Yurika; Tateishi, Sayuri

    2010-01-01

    It has been reported that oil heated with vegetable protein under reduced pressure, followed by filtration (soy oil), decreased body, liver and retroperitoneal fat tissue weights and serum triacylglycerol levels in Wistar rats. In order to clarify the mechanism of these weight-loss promoting effects, gastrointestinal tract content transfer was traced. Fasted 10-week-old rats were fed a slurry containing AIN93G without fat, Cr(2)O(3) (marker), and 7 wt% soy oil or fresh oil (control) and sacrificed at 20, 60, 120, or 360 min; then, blood, stomach, small intestine, cecum, colon and feces were collected. The results indicated that the content transferred faster from stomach to small intestine in the soy oil group than in the control group. At 60 min after the ingestion of diet, an increased serum triacylglycerol level was found in the soy oil group. In addition, fecal excretion in the soy oil group was significantly higher 120 min after the administration than in the control group, suggesting that soy oil stimulated peristalsis of the colon and that colon contents (food ingested before administration) were actively excreted.

  11. Amelogenin processing by MMP-20 prevents protein occlusion inside calcite crystals.

    Science.gov (United States)

    Bromley, Keith M; Lakshminarayanan, Rajamani; Thompson, Mitchell; Lokappa, Sowmya B; Gallon, Victoria A; Cho, Kang R; Qiu, S Roger; Moradian-Oldak, Janet

    2012-10-03

    Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.

  12. 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.

  13. 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.

  14. 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.

  15. Amino Acid and Mineral Supplementation in Fermentation Process of Concentrate Protein of Jatropha Seed Cake (Jatropha curcas L.

    Directory of Open Access Journals (Sweden)

    Titin Widiyastuti

    2016-09-01

    Full Text Available The purpose of this study is to assess the optimization of fermentation process by adding a minerals and amino acids so that the potential of protein  of Concentrate Protein-Jatropha seed cake (CP-JSC can be optimally used as a substitute for soybean meal. The method used was completely randomized design. The treatment consisted of F1: Fermentation CP-BBJ + methionine-lysine (0.25%: 0.25%, F2: Fermentation CP-JSC + methionine-lysine (0.5%: 0.5%, F3: F1 + 0.45% Dicalsium Phosphate, F4: F2 + 0.45% Dicalsium Phosphate. Each treatment was repeated four times, When treatment significantly continued by Least Significant Difference (LSD, variables observed are the levels of antinutrients (phorbolester, antitrypsin, the levels of nutrients (fat, protein, crude fiber, Ca, P and gross energy and amino acid. Results of analysis of variance showed that the addition of amino acids and minerals Ca, P in the fermentation process was highly significant effect on the levels of crude fiber and phosphorus (P 0.05. While the levels obtained phorbolester range of 0.055% - 0.08%. It was concluded that the optimization of fermentation can be done without adding the amino acid supplementation of minerals calcium and phosphorus. Supplementation significantly affect a significant increase or decrease in some nutrients (crude fiber, gross energy, phosphor and capable of suppressing a decrease in amino acids. Supplementation of amino acids Lysine and Methionin 0.05% is the best treatment.

  16. 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.

  17. Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants.

    Science.gov (United States)

    Halpin, C; Cooke, S E; Barakate, A; El Amrani, A; Ryan, M D

    1999-02-01

    Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co-ordinate expression of active CAT and GUS. Self-processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring co-ordinated, stable expression of multiple introduced proteins in plant cells.

  18. Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein.

    Directory of Open Access Journals (Sweden)

    Entsar Saheb

    2015-06-01

    Full Text Available Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host's immune response. Acanthamoeba Type-I metacaspase (Acmcp is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein.The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr, were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr.Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings.Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii.

  19. The yeast SR-like protein Npl3 links chromatin modification to mRNA processing.

    Directory of Open Access Journals (Sweden)

    Erica A Moehle

    Full Text Available Eukaryotic gene expression involves tight coordination between transcription and pre-mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA-independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre-mRNA splicing.

  20. 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.

  1. 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

    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...... 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...

  2. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Philip W. Brownjohn

    2017-04-01

    Full Text Available Summary: Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP processing and the accumulation of APP-derived amyloid β (Aβ peptides are key processes in Alzheimer's disease (AD. We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation. : In this article, Livesey and colleagues perform a phenotypic drug screen in a human stem cell model of Alzheimer's disease. The anthelminthic avermectins are identified as a family of compounds that increase the production of short Aβ peptides over longer more toxic Aβ forms. The effect is analogous to existing γ-secretase modulators, but is independent of the core γ-secretase complex. Keywords: neural stem cells, Alzheimer's disease, phenotypic screening, iPSCs, human neurons, dementia, Down syndrome, amyloid beta, ivermectin, selamectin

  3. Using protein nanofibrils to remove azo dyes from aqueous solution by the coagulation process.

    Science.gov (United States)

    Morshedi, Dina; Mohammadi, Zeinab; Akbar Boojar, Masoud Mashhadi; Aliakbari, Farhang

    2013-12-01

    The ever-increasing applications of hazardous azo dyes as industrialized coloring agents have led to serious remediation challenges. In this study, proteinaceous nanofibrils were examined as coagulants for decolorization of azo dyes in aqueous solutions. The results provided some insight regarding the mechanism of dye removal. The strength of nanofibrils to remove dyes from solution was evaluated by remediation of acid red 88, Bismarck brown R, direct violet 51, reactive black 5, and Congo red. However, the efficiency of nanofibrils to coagulate with different dyes was variable (60-98%) and dependent on the structures of dyes and the physicochemical conditions of the solutions. Increasing the temperature or ionic strength declined the coagulation time and induced the rate of dye removal. Changing pH had contradictory effects on the dye removal efficiency which was more affected by the chemical structure of the dye rather than the change in stability of the coagulant. The efficiency of nanofibrils to remove dyes was more than that of charcoal, which is considered as one of the most common substances used for azo dye remediation which may be due to its well dispersion in the aqueous solutions, and slower rates of the coagulation than that of the adsorption process. Furthermore, cytotoxicity was not detected after treating cell cultures with the decolorized solutions. Accordingly, by integrating biological and biophysicochemical processes, proteinaceous nanofibrils can be promising candidates for treatment of colored wastewaters. Ease of production, proper and quick dispersion in water, without the production of dangerous dye by-products and derivatives, are some of the main advantages of nanofibrils. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. The use of glycohydrolase in the processing of hull-less seed variety of pumpkin in the relation to enhanced protein extraction

    Directory of Open Access Journals (Sweden)

    Peričin Draginja M.

    2006-01-01

    Full Text Available Enzyme hydrolysis optimisation of cell-wall polysaccharides of pumpkin seed with celluloses and pectinases and their mixture, in the relation to enhanced protein extraction was the objective of this study. The individual and combined effects of cell-wall-degrading enzyme activities on protein extraction combined with other process parameters: enzyme concentration and time of hydrolysis were evaluated by the Response Surface Methodology (RSM. The optimal value protein isolate yield (9 g of soluble protein from J 00 g pumpkin seed is achieved under the following conditions: 2% enzymes (celluloses, reaction time 300 min, pH = 5.0, temperature 45°C.

  5. MERISTEM DISORGANIZATION1 encodes TEN1, an essential telomere protein that modulates telomerase processivity in Arabidopsis.

    Science.gov (United States)

    Leehy, Katherine A; Lee, Jung Ro; Song, Xiangyu; Renfrew, Kyle B; Shippen, Dorothy E

    2013-04-01

    Telomeres protect chromosome ends from being recognized as DNA damage, and they facilitate the complete replication of linear chromosomes. CST [for CTC1(Cdc13)/STN1/TEN1] is a trimeric chromosome end binding complex implicated in both aspects of telomere function. Here, we characterize TEN1 in the flowering plant Arabidopsis thaliana. We report that TEN1 (for telomeric pathways in association with Stn1, which stands for suppressor of cdc thirteen) is encoded by a previously characterized gene, MERISTEM DISORGANIZATION1 (MDO1). A point mutation in MDO1, mdo1-1/ten1-3 (G77E), triggers stem cell differentiation and death as well as a constitutive DNA damage response. We provide biochemical and genetic evidence that ten1-3 is likely to be a null mutation. As with ctc1 and stn1 null mutants, telomere tracts in ten1-3 are shorter and more heterogeneous than the wild type. Mutants also exhibit frequent telomere fusions, increased single-strand telomeric DNA, and telomeric circles. However, unlike stn1 or ctc1 mutants, telomerase enzyme activity is elevated in ten1-3 mutants due to an increase in repeat addition processivity. In addition, TEN1 is detected at a significantly smaller fraction of telomeres than CTC1. These data indicate that TEN1 is critical for telomere stability and also plays an unexpected role in modulating telomerase enzyme activity.

  6. 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.

  7. The membrane separation mechanism in protein concentration from the extract of waste press cake in biofuel manufacturing process of Jatropha seeds

    Science.gov (United States)

    Chung, T. W.; Chen, C. K.; Hsu, S. H.

    2017-11-01

    Protein concentration process using filter membrane has a significant advantage on energy saving compared to the traditional drying processes. However, fouling on large membrane area and frequent membrane cleaning will increase the energy consumption and operation cost for the protein concentration process with filter membrane. In this study, the membrane filtration for protein concentration will be conducted and compared with the recent protein concentration technology. The analysis of operating factors for protein concentration process using filter membrane was discussed. The separation mechanism of membrane filtration was developed according to the size difference between the pore of membrane and the particle of filter material. The Darcy’s Law was applied to discuss the interaction on flux, TMP (transmembrane pressure) and resistance in this study. The effect of membrane pore size, pH value and TMP on the steady-state flux (Jst) and protein rejection (R) were studied. It is observed that the Jst increases with decreasing membrane pore size, the Jst increases with increasing TMP, and R increased with decreasing solution pH value. Compare to other variables, the pH value is the most significant variable for separation between protein and water.

  8. 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.

  9. 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

  10. 17-AAG improves cognitive process and increases heat shock protein response in a model lesion with Aβ25-35.

    Science.gov (United States)

    Ortega, Laura; Calvillo, Minerva; Luna, Félix; Pérez-Severiano, Francisca; Rubio-Osornio, Moisés; Guevara, Jorge; Limón, Ilhuicamina Daniel

    2014-08-01

    Molecular chaperones, or heat shock proteins (HSP), have been implicated in numerous neurodegenerative disorders characterized by the accumulation of protein aggregates, such as Alzheimer disease. The agglomeration of insoluble structures of Aβ is thought to be responsible for neuronal death, which in turn leads to the loss of cognitive functions. Recent findings have shown that the induction of HSP decreases the level of abnormal protein aggregates, as well as demonstrating that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an analogue of geldanamycin (GA), increases Aβ clearance through the induction of molecular chaperones in cell culture. In light of this discovery that HSP overexpression can be neuroprotective, the search for a way to pharmacologically induce the overexpression of HSP and other associated chaperones may lead to a promising approach for the treatment of neurodegenerative diseases. The aim of our study was to evaluate both the effect of 17-AAG on the cognitive process and the HSP response in rats injected with Aβ25-35 into the CA1 of the hippocampus. The results show that the injection of Aβ caused a significant increase in the expression of the HSP involved in the regulation of cellular proteostasis. While the HSP did not reverse excitotoxic damage, given that experimental subjects showed learning and memory deficits, the administration of 17-AAG prior to the injection of Aβ25-35 did show an improvement in the behavioral assessment that correlated with the upregulation of HSP70 in subjects injured with Aβ. Overall, our data shows that the pharmacological induction of HSP using 17-AAG may be an alternative treatment of neurodegenerative diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Effects of different industrial heating processes of milk on site-specific protein modifications and their relationship to in vitro and in vivo digestibility.

    Science.gov (United States)

    Wada, Yasuaki; Lönnerdal, Bo

    2014-05-07

    Heating processes are applied to milk and dairy products to ensure their microbiological safety and shelf lives. However, how differences in "industrial" thermal treatments affect protein digestibility is still equivocal. In this study, raw milk was subjected to pasteurization, three kinds of ultra-high-temperature (UHT) treatment, and in-can sterilization and was investigated by in vitro and in vivo digestion and proteomic methods. In-can sterilized milk, followed by UHT milk samples, showed a rapid decrease in protein bands during the course of digestion. However, protein digestibility determined by a Kjeldahl procedure showed insignificant differences. Proteomic analysis revealed that lactulosyllysine, which reflects a decrease in protein digestibility, in α-lactalbumin, β-lactoglobulin, and caseins was higher in in-can sterilized milk, followed by UHT milk samples. Thus, industrial heating may improve the digestibility of milk proteins by denaturation, but the improvement is likely to be offset by heat-derived modifications involved in decreased protein digestibility.

  12. A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins.

    Science.gov (United States)

    Xu, Weichen; Jimenez, Rod Brian; Mowery, Rachel; Luo, Haibin; Cao, Mingyan; Agarwal, Nitin; Ramos, Irina; Wang, Xiangyang; Wang, Jihong

    2017-10-01

    During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.

  13. Sensitizing and Eliciting Capacity of Egg White Proteins in BALB/c Mice As Affected by Processing.

    Science.gov (United States)

    Pablos-Tanarro, Alba; Lozano-Ojalvo, Daniel; Martínez-Blanco, Mónica; López-Fandiño, Rosina; Molina, Elena

    2017-06-07

    This study assesses to what extent technological processes that lead to different degrees of denaturation of egg white proteins affect their allergenicity. We focused on heat (80 °C, 10 min) and high-pressure (400 MPa and 37 °C, 10 min) treatments and used a BALB/c mouse model of food allergy. Oral sensitization to egg white using cholera toxin as adjuvant induced the production of IgE and IgG1 isotypes and led to severe clinical signs following challenge with the allergen. Extensive protein denaturation caused by heat treatment increased its ability to induce Th1 responses and reduced both its sensitizing and eliciting capacity. Heated egg white stimulated the production of IgE over IgG1 antibodies directed, at least in part, toward new epitopes exposed as a result of heat treatment. Conversely, partial denaturation caused by high-pressure treatment increased the ability of egg white to stimulate Th2 responses and its allergenic potential.

  14. Influence of sorbitol on mechanical and physico-chemical properties of soy protein-based bioplastics processed by injection molding

    Directory of Open Access Journals (Sweden)

    Manuel Felix

    Full Text Available Abstract Soy Protein Isolate (SPI has been evaluated as useful candidate for the development of protein-based bioplastic materials processed by injection molding. The influence of sorbitol (SB as plasticizer in mechanical properties and water uptake capacity was evaluated in SPI-based bioplastics. A mixing rheometer that allows monitoring torque and temperature during mixing and a small-scale-plunger-type injection molding machine were used to obtain SPI/Plasticizer blends and SPI-based bioplastics, respectively. Dynamic measurements were carried out to obtain mechanical spectra of different bioplastics. Moreover, the mechanical characterization was supplemented with uniaxial tensile tests. Additionally, the influence of SB in water uptake capacity was also evaluated. The introduction of SB leads to increase the rigidity of bioplastics as well as the water uptake capacity after 24h, however it involves a decrease in strain at break. Final bioplastics are plastic materials with both adequate properties for the substitution of conventional petroleum plastics and high biodegradability.

  15. 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.

  16. 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

    Incubation of Fe(III)myoglobin (Fe(III)Mb) with H2O2 in the presence of bovine serum albumin (BSA) has been shown previously to give albumin-derived radicals as a result of radical transfer from myoglobin to BSA. In this study the occurrence of similar processes with peroxidases has been...... investigated using horseradish peroxidase (HRP)/H2O2, in the presence and absence of added tyrosine. Incubation of HRP with H2O2 and bovine or human serum albumins, in the presence and absence of tyrosine, gave long-lived albumin-derived radicals as detected by EPR spectroscopy. Evidence has been obtained...... for these albumin radicals being located on buried tyrosine residues on the basis of blocking experiments. The effect of protein conformation on radical transfer has been investigated using partial proteolytic digestion prior to protein oxidation. With HRP/H2O2/BSA and Fe(III)Mb/H2O2/BSA increased radical...

  17. 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.

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

    Science.gov (United States)

    Peruquetti, Rita L; de Mateo, Sara; Sassone-Corsi, Paolo

    2012-01-01

    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.

  19. Nutritional and Digestive Properties of Protein Isolates Extracted from the Muscle of the Common Carp Using pH-Shift Processing.

    Science.gov (United States)

    Tian, Yuanyong; Wang, Wei; Yuan, Chunhong; Zhang, Long; Liu, Jinyang; Liu, Junrong

    2017-02-01

    This study details the nutritional and digestive properties of protein isolates that are extracted from carp ( Cyprinus Carpio L.) muscle using pH shifting methods. Alkaline (ALPI) and acid (ACPI) protein isolates exhibit higher protein yields (87.6%, 76.3%, respectively). In addition to the high recovery of myofibrillar protein, a portion of the water-soluble proteins is also recovered. The moisture contents of ACPI and ALPI are 85.5% and 88.5%, respectively, and the crude protein contents of these two fractions are 83.20% and 83.0%, respectively, both contents of which are higher than those for fresh muscle. Most part of the ash and fat are removed in the separation process. The protein isolation is also found to be lighter and whiter than the fresh muscle and there is no difference between amino acid content of protein isolation and that of fresh muscle. The maximum solubility of water washed surimi is 73.21%, while solubility of ACPI-2 and ALPI-2 (pH 7.0) are 66.67% and 62.08%, respectively. The digestibility of ALPI and ACPI is improved after being treated with chymotrypsin, which is about 7-8 times as that of fresh muscle. The results indicate that the protein isolates have better nutritional and digestive properties than the fresh muscle does in food processing. Common carp is a lower additional value fish that exists in large amount in China. This study investigates nutritional and digestive properties of protein from carp extracted by pH shifting methods. According to the obtained data in this study, pH shifting method is a good protein recovery method that can effectively remove bone spurs, skin, fat and other impurities. In addition, sarcoplasmic proteins can also be recovered. The nutritional properties of protein isolates of carp were suitable for supplementing as an ingredient for human consumption. The pH-shift process greatly improves the protein digestibility. Therefore, there are broad application prospects of the protein isolation as protein

  20. Euglena in time: Evolution, control of central metabolic processes and multi-domain proteins in carbohydrate and natural product biochemistry

    Directory of Open Access Journals (Sweden)

    Ellis C. O’Neill

    2015-12-01

    Full Text Available Euglena gracilis is a eukaryotic microalgae that has been the subject of scientific study for hundreds of years. It has a complex evolutionary history, with traces of at least four endosymbiotic genomes and extensive horizontal gene transfer. Given the importance of Euglena in terms of evolutionary cell biology and its unique taxonomic position, we initiated a de novo transcriptome sequencing project in order to understand this intriguing organism. By analysing the proteins encoded in this transcriptome, we can identify an extremely complex metabolic capacity, rivalling that of multicellular organisms. Many genes have been acquired from what are now very distantly related species. Herein we consider the biology of Euglena in different time frames, from evolution through control of cell biology to metabolic processes associated with carbohydrate and natural products biochemistry.

  1. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein.

    Science.gov (United States)

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-11-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackievirus B3 (CVB3), substitution N2D in 2C was identified in each of the PI4KB-inhibitor resistant CVB3 pools, but its possible benefit has not been investigated yet. In this study, we set out to investigate the possible role of 2C-N2D in the resistance to PI4KB and OSBP inhibition. We show that 2C-N2D by itself did not confer any resistance to inhibitors of PI4KB and OSBP. However, the double mutant (i.e., 2C-N2D/3A-H57Y) showed better replication than the 3A-H57Y single mutant in the presence of inhibitors. Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the poliovirus polyprotein. Therefore, we studied the effect of PI4KB or OSBP inhibition on proteolytic processing of the CVB3 polyprotein during infection as well as in a replication-independent system. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y recovered impaired proteolytic processing at this junction. Although 2C-N2D enhanced replication of the 3A-H57Y single mutant, we did not detect additional effects of this substitution on polyprotein processing, which leaves the mechanism of how 2C-N2D contributes to the resistance to be revealed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

    Science.gov (United States)

    Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

    2018-01-15

    Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

  3. 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.

  4. A new data processing routine facilitating the identification of surface adhered proteins from bacterial conditioning films via QCM-D/MALDI-ToF/MS.

    Science.gov (United States)

    Hohmann, Siegfried; Neidig, Anke; Kühl, Boris; Kirschhöfer, Frank; Overhage, Jörg; Brenner-Weiß, Gerald

    2017-10-01

    Conditioning films are an important factor in the initiation and development of microbial biofilms, which are the leading cause of chronic infections associated with medical devices. Here, we analyzed the protein content of conditioning films formed after exposure to supernatants of cultures of the human pathogen Pseudomonas aeruginosa PAO1. Adhesion of substances from the supernatant was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) sensor chips modified with the commonly used implant material titanium dioxide (TiO 2 ). Attached proteins were identified after on-chip digestion using matrix-assisted laser desorption/ionization (MALDI) time of flight (ToF) mass spectrometry (MS), and a new data processing tool consisting of an XML-database with theoretical tryptic peptides of every PAO1 protein and PHP scripts. Sub-databases containing only proteins, that we found in all replicates, were created and used for MS/MS precursor selection. The obtained MS/MS peaklists were then matched against theoretical fragmentations of the expected peptide sequences to verify protein identification. Using this approach we were able to identify 40 surface-associated proteins. In addition to extracellular proteins such as adhesins, a number of intra-cellular proteins were identified which may be involved in conditioning film formation, suggesting an as-yet unidentified role for these proteins, possibly after cell lysis. Graphical Abstract Flowchart of the method.

  5. The Orosomucoid 1 protein is involved in the vitamin D – mediated macrophage de-activation process

    International Nuclear Information System (INIS)

    Gemelli, Claudia; Martello, Andrea; Montanari, Monica; Zanocco Marani, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis

    2013-01-01

    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

  6. 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.

  7. 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

    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 repli...

  8. 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.

  9. 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.

  10. Simultaneous determination of the protein conversion process in porcine stratum corneum after pretreatment with skin enhancers by a combined microscopic FT-IR/DSC system

    Science.gov (United States)

    Lin, Shan-Yang; Duan, Kwo-Jen; Lin, Tsung-Chien

    1996-11-01

    A newly developed microscopic Fourier transform infrared (FT-IR) spectrometry combined with differential scanning calorimetry (DSC) has been used to investigate simultaneously the thermal response and IR spectral changes in protein structure in porcine stratum corneum (SC) after pretreatment with skin penetration enhancers (propylene glycol (PG), azone/PG, oleic acid (OA)/PG, vitamin C, and vitamin C+ OA/PG). The amide I and II bands of the protein were used as probes to determine its structural transformation with temperature. A reheating process was also performed. The dual effects of enhancer and temperature on the protein conformational changes of porcine SC were studied. The results indicate that the newly developed FT-IR/DSC system can continuously determine the thermoresponsive conversion process from α-helix to β-sheet in the keratin structure of porcine SC pretreated with different enhancers. The temperature-induced keratin conversion in the protein structure of porcine SC was irreversible, with or without pretreatment with skin penetration enhancers. The conformational transition in the protein during heating was found to be partially from the α-helix to a random coil structure, and partially from the α-helix to the β-sheet structure. The kinetics of this conversion for the first and second heating processes were significantly different; the conversion process for all the first-heated SC samples during the second heating process was slower than that of the samples during the first heating process. Moreover, it was found that the skin penetration enhancers were able to alter synergistically and promote keratin conversion in the protein structure of porcine SC when accompanying the heating process. PG, OA/PG and azone/PG were found to be the most effective.

  11. Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar

    Directory of Open Access Journals (Sweden)

    Adriana Cheavegatti-Gianotto

    2018-03-01

    Full Text Available Brazil is the largest sugarcane producer and the main sugar exporter in the world. The industrial processes applied by Brazilian mills are very efficient in producing highly purified sugar and ethanol. Literature presents evidence of lack of DNA/protein in these products, regardless of the nature of sugarcane used as raw material. Recently CTNBio, the Brazilian biosafety authority, has approved the first biotechnology-derived sugarcane variety for cultivation, event CTC175-A, which expresses the Cry1Ab protein to control the sugarcane borer (Diatraea saccharalis. The event also expresses neomycin-phosphotransferase type II (NptII protein used as selectable marker during the transformation process. Because of the high purity of sugar and ethanol produced from genetically modified sugarcane, these end-products should potentially be classified as “pure substances, chemically defined,” by Brazilian Biosafety Law No. 11.105. If this classification is to be adopted, these substances are not considered as “GMO derivatives” and fall out of the scope of Law No. 11.105. In order to assess sugar composition and quality, we evaluate Cry1Ab and NptII expression in several sugarcane tissues and in several fractions from laboratory-scale processing of event CTC175-A for the presence of these heterologous proteins as well as for the presence of traces of recombinant DNA. The results of these studies show that CTC175-A presents high expression of Cry1Ab in leaves and barely detectable expression of heterologous proteins in stalks. We also evaluated the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase protein and DNA in the fractions of the industrial processing of conventional Brazilian sugarcane cultivars. Results from both laboratory and industrial processing were concordant, demonstrating that DNA and protein are not detected in the clarified juice and downstream processed fractions, including ethanol and raw sugar, indicating that protein

  12. Most consumed processed foods by patients on hemodialysis: Alert for phosphate-containing additives and the phosphate-to-protein ratio.

    Science.gov (United States)

    Watanabe, Marcela T; Araujo, Raphael M; Vogt, Barbara P; Barretti, Pasqual; Caramori, Jacqueline C T

    2016-08-01

    Hyperphosphatemia is common in patients with chronic kidney disease (CKD) stages IV and V because of decreased phosphorus excretion. Phosphatemia is closely related to dietary intake. Thus, a better understanding of sources of dietary phosphate consumption, absorption and restriction, particularly inorganic phosphate found in food additives, is key to prevent consequences of this complication. Our aims were to investigate the most commonly consumed processed foods by patients with CKD on hemodialysis, to analyze phosphate and protein content of these foods using chemical analysis and to compare these processed foods with fresh foods. We performed a cross-sectional descriptive analytical study using food frequency questionnaires to rank the most consumed industrialized foods and beverages. Total phosphate content was determined by metavanadate colorimetry, and nitrogen content was determined by the Kjeldahl method. Protein amounts were estimated from nitrogen content. The phosphate-to-protein ratio (mg/g) was then calculated. Processed meat protein and phosphate content were compared with the nutritional composition of fresh foods using the Brazilian Food Composition Table. Phosphate measurement results were compared with data from the Food Composition Table - Support for Nutritional Decisions. An α level of 5% was considered significant. Food frequency questionnaires were performed on 100 patients (mean age, 59 ± 14 years; 57% male). Phosphate additives were mentioned on 70% of the product labels analyzed. Proteins with phosphate-containing additives provided approximately twice as much phosphate per gram of protein compared with that of fresh foods (p processed foods are higher than those of fresh foods, as well as phosphate-to-protein ratio. A better understanding of phosphate content in foods, particularly processed foods, may contribute to better control of phosphatemia in patients with CKD. Copyright © 2016 European Society for Clinical Nutrition and

  13. Detect the sensitivity and response of protein molecular structure of whole canola seed (yellow and brown) to different heat processing methods and relation to protein utilization and availability using ATR-FT/IR molecular spectroscopy with chemometrics

    Science.gov (United States)

    Samadi; Theodoridou, Katerina; Yu, Peiqiang

    2013-03-01

    The objectives of this experiment were to detect the sensitivity and response of protein molecular structure of whole canola seed to different heat processing [moisture (autoclaving) vs. dry (roasting) heating] and quantify heat-induced protein molecular structure changes in relation to protein utilization and availability. In this study, whole canola seeds were autoclaved (moisture heating) and dry (roasting) heated at 120 °C for 1 h, respectively. The parameters assessed included changes in (1) chemical composition profile, (2) CNCPS protein subfractions (PA, PB1, PB2, PB3, PC), (3) intestinal absorbed true protein supply, (4) energy values, and (5) protein molecular structures (amide I, amide II, ratio of amide I to II, α-helix, β-sheet, ratio of α-helix to β-sheet). The results showed that autoclave heating significantly decreased (P canola seed. Future study is needed to study response and impact of heat processing to each inherent layer of canola seed from outside to inside tissues and between yellow canola and brown canola.

  14. 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.

  15. Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris.

    Science.gov (United States)

    Maurer, Michael; Kühleitner, Manfred; Gasser, Brigitte; Mattanovich, Diethard

    2006-12-11

    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. 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. 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.

  16. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Mass Spectrometry-based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds.

    Science.gov (United States)

    Steinhilber, Andreas E; Schmidt, Felix F; Naboulsi, Wael; Planatscher, Hannes; Niedzwiecka, Alicia; Zagon, Jutta; Braeuning, Albert; Lampen, Alfonso; Joos, Thomas O; Poetz, Oliver

    2018-02-22

    The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR), are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of non-ruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides and a LC-MS/MS based analysis for peptide quantification using isotope labelled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of four orders of magnitude with a detection limit in the range of 0.1 % to 1 % (w/w).

  18. 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. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Modelling the metabolic characteristics of proteins in dairy cattle from co-products of bioethanol processing: comparison of the NRC 2001 model with the DVE/OEB system.

    Science.gov (United States)

    Nuez-Ortín, Waldo G; Yu, Peiqiang

    2011-02-01

    Co-products from bioethanol processing include wheat dried distillers grains with solubles (DDGS), corn DDGS, blend DDGS (e.g. wheat/corn at 70:30, 60:40 or 50:50 w/w), triticale DDGS, barley DDGS and pea DDGS. The objective of this study was to compare two systems, the DVE/OEB system versus the NRC 2001 model, in modelling the metabolic characteristics of proteins in dairy cattle from different types of co-products (DDGS) from different bioethanol processing plants. The predicted values from the NRC 2001 model were 10% higher (P 0.05). The sensitivity of the two models in detecting differences among DDGS types and between bioethanol plants was similar. The two models coincided in the superior protein value of blend DDGS as well as in the more optimal degraded protein balance (DPB) for corn DDGS. Although the differences between the DVE/OEB system and the NRC 2001 model were significant (P < 0.05) for most outputs owing to differences in some of the concepts and factors used in modelling, the correlations between total truly absorbed protein (DVE) and metabolisable protein (MP) values and between degraded protein balances (DPB(OEB) vs DPB(NRC) ) were also significant (P < 0.05). 2010 Society of Chemical Industry.

  20. Trafficking in Alzheimer's Disease: Modulation of APP Transport and Processing by the Transmembrane Proteins LRP1, SorLA, SorCS1c, Sortilin, and Calsyntenin.

    Science.gov (United States)

    Eggert, Simone; Thomas, Carolin; Kins, Stefan; Hermey, Guido

    2017-10-27

    The amyloid precursor protein (APP), one key player in Alzheimer's disease (AD), is extensively processed by different proteases. This leads to the generation of diverging fragments including the amyloid β (Aβ) peptide, which accumulates in brains of AD patients. Subcellular trafficking of APP is an important aspect for its proteolytic conversion, since the various secretases which cleave APP are located in different cellular compartments. As a consequence, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The mechanisms underlying intracellular APP transport are critical to understand AD pathogenesis and can serve as a target for future pharmacological interventions. In the recent years, a number of APP interacting proteins were identified which are implicated in sorting of APP, thereby influencing APP processing at different angles of the secretory or endocytic pathway. This review provides an update on the proteolytic processing of APP and the interplay of the transmembrane proteins low-density lipoprotein receptor-related protein 1, sortilin-receptor with A-type repeats, SorCS1c, sortilin, and calsyntenin. We discuss the specific interactions with APP, the capacity to modulate the intracellular itinerary and the proteolytic conversion of APP, a possible involvement in the clearance of Aβ, and the implications of these transmembrane proteins in AD and other neurodegenerative diseases.

  1. Ferrous iron oxidation by sulfur-oxidizing Acidithiobacillus ferrooxidans and analysis of the process at the levels of transcription and protein synthesis.

    Science.gov (United States)

    Kucera, Jiri; Bouchal, Pavel; Lochman, Jan; Potesil, David; Janiczek, Oldrich; Zdrahal, Zbynek; Mandl, Martin

    2013-04-01

    In contrast to iron-oxidizing Acidithiobacillus ferrooxidans, A. ferrooxidans from a stationary phase elemental sulfur-oxidizing culture exhibited a lag phase in pyrite oxidation, which is similar to its behaviour during ferrous iron oxidation. The ability of elemental sulfur-oxidizing A. ferrooxidans to immediately oxidize ferrous iron or pyrite without a lag phase was only observed in bacteria obtained from growing cultures with elemental sulfur. However, these cultures that shifted to ferrous iron oxidation showed a low rate of ferrous iron oxidation while no growth was observed. Two-dimensional gel electrophoresis was used for a quantitative proteomic analysis of the adaptation process when bacteria were switched from elemental sulfur to ferrous iron. A comparison of total cell lysates revealed 39 proteins whose increase or decrease in abundance was related to this phenotypic switching. However, only a few proteins were closely related to iron and sulfur metabolism. Reverse-transcription quantitative PCR was used to further characterize the bacterial adaptation process. The expression profiles of selected genes primarily involved in the ferrous iron oxidation indicated that phenotypic switching is a complex process that includes the activation of genes encoding a membrane protein, maturation proteins, electron transport proteins and their regulators.

  2. Freeze-Drying of L-Arginine/Sucrose-Based Protein Formulations, Part 2: Optimization of Formulation Design and Freeze-Drying Process Conditions for an L-Arginine Chloride-Based Protein Formulation System.

    Science.gov (United States)

    Stärtzel, Peter; Gieseler, Henning; Gieseler, Margit; Abdul-Fattah, Ahmad M; Adler, Michael; Mahler, Hanns-Christian; Goldbach, Pierre

    2015-12-01

    We recently reported that the presence of chloride counter ions in freeze-dried l-arginine/sucrose formulations provided the greatest protein stability, but led to low collapse temperatures and glass transition temperatures of the freeze concentrates. The objectives of this study were to identify l-arginine chloride-based formulations and optimize freeze-drying process conditions to deliver a freeze-dried product with good physical quality attributes (including cake appearance, residual moisture, and reconstitution time). Additional properties were tested such as thermal properties, cake microstructure, and protein physical stability. Excipient concentrations were varied with and without a model protein (bovine serum albumin, BSA). Formulations were frozen with and without annealing or with and without controlled nucleation. Primary drying was conducted at high and low shelf temperature. Cakes with least defects and optimum physical attributes were achieved when protein to excipient ratios were high. Controlled nucleation led to elegant cakes for most systems at a low shelf temperature. Replacing BSA by a monoclonal antibody showed that protein (physical) stability was slightly improved under stress storage temperature (i.e., 40°C) in the presence of a low concentration of l-arginine in a sucrose-based formulation. At higher l-arginine concentrations, cake defects increased. Using optimized formulation design, addition of l-arginine chloride to a sucrose-based formulation provided elegant cakes and benefits for protein stability. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  3. The effect of Polyscias filicifolia bailey biomass tincture on the protein synthesis process in the heterogeneous system from the isolated pig heart.

    Science.gov (United States)

    Kašauskas, Artūras; Mongirdienė, Aušra

    2013-01-01

    An insufficient supply of oxygen to the heart influences the process of protein synthesis. The aim of this study was to determine the effect of the Polyscias filicifolia Bailey biomass tincture on the protein synthesis process in a heterogeneous translation system from the isolated pig heart. The effect of anoxia was evaluated after 20- and 90-minute anoxia. With the aim to determine the effect of Polyscias, the pig hearts were perfused with a buffer containing the Polyscias filicifolia Bailey biomass tincture. To determine the rate and the level of translation, the incorporation of [(14)C]-leucine into translational products in a cell-free system was measured. The protein synthesis level decreased by 23%-42% when the translation system containing cytosol from the anoxic heart was used. When the translation system containing a ribosomal fraction after 20-minutes anoxia was used, the protein synthesis level was the same as in the control. In the case of 90-minute anoxia, it decreased by 16%. The protein synthesis rate and the level in the translation system containing cytosol from the heart after 20-minute anoxic perfusion with the buffer containing Polyscias was the same as in the control. A decrease in the protein synthesis rate and the level after 20-minute anoxia was determined by changes in cytosol. On the other hand, 90-minute anoxia caused changes in cytosol and the ribosomal fraction. The Polyscias filicifolia Bailey biomass tincture restored the protein synthesis process acting on the components of the translation system in cytosol and the ribosomal fraction.

  4. Modeling and computations of the intramolecular electron transfer process in the two-heme protein cytochrome c4

    DEFF Research Database (Denmark)

    Natzmutdinov, Renat R.; Bronshtein, Michael D.; Zinkicheva, Tamara T.

    2012-01-01

    The di-heme protein Pseudomonas stutzeri cytochrome c4 (cyt c4) has emerged as a useful model for studying long-range protein electron transfer (ET). Recent experimental observations have shown a dramatically different pattern of intramolecular ET between the two heme groups in different local...

  5. Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome

    DEFF Research Database (Denmark)

    Feizi, Amir; Gatto, Francesco; Uhlén, Mathias

    2017-01-01

    Protein secretory pathway in eukaryal cells is responsible for delivering functional secretory proteins. The dysfunction of this pathway causes a range of important human diseases from congenital disorders to cancer. Despite the piled-up knowledge on the molecular biology and biochemistry level...

  6. Procédé IFP de production de protéines ex-méthanol Ifp Process for Protein Production Ex Methanol

    OpenAIRE

    Ballerini D.

    2006-01-01

    Dans cet article, on expose les principales caractéristiques du procédé IFP de production de protéines ex-méthanol en les comparant à celles du procédé à partir de n-paraffines. This article describes the main features of the IFP process for producing proteins ex-methanol by comparing them with those of the process ex n-paraffins.

  7. Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

    Science.gov (United States)

    Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

    2014-07-16

    We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence

  8. Doublecortin-like, a microtubule-associated protein expressed in radial glia, is crucial for neuronal precursor division and radial process stability.

    NARCIS (Netherlands)

    Vreugdenhil, E.; Kolk, S.H.; Boekhoorn, K.; Fitzsimons, C.P.; Schaaf, M; Schouten, Th.; Sarabdjitsingh, A.; Sibug, R.; Lucassen, P.J.

    2007-01-01

    During corticogenesis, progenitors divide within the ventricular zone where they rely on radial process extensions, formed by radial glial cell (RG) scaffolds, along which they migrate to the proper layers of the cerebral cortex. Although the microtubule-associated proteins doublecortin (DCX) and

  9. In vitro assay for the Bacillus subtilis signal peptidase SipS : systems for efficient in vitro transcription-translation and processing of precursors of secreted proteins

    NARCIS (Netherlands)

    VEHMAANPERA, J; GORNER, A; VENEMA, G; BRON, S; van Dijl, Jan Maarten

    1993-01-01

    The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins. It differs from the SPases of Gram-negative bacteria in structure and specificity. To assay the activity of SipS in vitro, two efficient transcription-translation systems for

  10. Continuous processing of recombinant proteins: integration of refolding and purification using simulated moving bed size-exclusion chromatography with buffer recycling.

    Science.gov (United States)

    Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

    2014-04-11

    Continuous processing of recombinant proteins was accomplished by combining continuous matrix-assisted refolding and purification by tandem simulated moving bed (SMB) size-exclusion chromatography (SEC). Recombinant proteins, N(pro) fusion proteins from inclusion bodies were dissolved with NaOH and refolded in the SMB system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling of the refolding buffer of the raffinate by tangential flow filtration. For further purification of the refolded proteins, a second SMB operation also based on SEC was added. The whole system could be operated isocratically with refolding buffer as the desorbent buffer, and buffer recycling could also be applied in the purification step. Thus, a significant reduction in buffer consumption was achieved. The system was evaluated with two proteins, the N(pro) fusion pep6His and N(pro) fusion MCP-1. Refolding solution, which contained residual N(pro) fusion peptide, the cleaved autoprotease N(pro), and the cleaved target peptide was used as feed solution. Full separation of the cleaved target peptide from residual proteins was achieved at a purity and recovery in the raffinate and extract, respectively, of approximately 100%. In addition, more than 99% of the refolding buffer of the raffinate was recycled. A comparison of throughput, productivity, and buffer consumption of the integrated continuous process with two batch processes demonstrated that up to 60-fold higher throughput, up to 180-fold higher productivity, and at least 28-fold lower buffer consumption can be obtained by the integrated continuous process, which compensates for the higher complexity. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Liquid-liquid phase separation causes high turbidity and pressure during low pH elution process in Protein A chromatography.

    Science.gov (United States)

    Luo, Haibin; Lee, Nacole; Wang, Xiangyang; Li, Yuling; Schmelzer, Albert; Hunter, Alan K; Pabst, Timothy; Wang, William K

    2017-03-10

    Turbid elution pools and high column back pressure are common during elution of monoclonal antibodies (mAbs) by acidic pH in Protein A chromatography. This phenomenon has been historically attributed to acid-induced precipitation of incorrectly folded or pH-sensitive mAbs and host cell proteins (HCPs). In this work, we propose a new mechanism that may account for some observations of elution turbidity in Protein A chromatography. We report several examples of turbidity and high column back pressure occurring transiently under a short course of neutral conditions during Protein A elution. A systematic study of three mAbs displaying this behavior revealed phase separation characterized by liquid drops under certain conditions including neutral pH, low ionic strength, and high protein concentration. These liquid droplets caused solution turbidity and exhibited extremely high viscosity, resulting in high column back pressure. We found out that the droplets were formed through liquid-liquid phase separation (LLPS) as a result of protein self-association. We also found multiple factors, including pH, temperature, ionic strength, and protein concentration can affect LLPS behaviors. Careful selection of process parameters during protein A elution, including temperature, flow rate, buffer, and salt can inhibit formation of a dense liquid phase, reducing both turbidity (by 90%) and column back pressure (below 20 pounds per square inch). These findings provide both mechanistic insight and practical mitigation strategies for Protein A chromatography induced LLPS. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  12. Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process

    Science.gov (United States)

    2014-01-01

    Background The chicken eggshell is a natural mechanical barrier to protect egg components from physical damage and microbial penetration. Its integrity and strength is critical for the development of the embryo or to ensure for consumers a table egg free of pathogens. This study compared global gene expression in laying hen uterus in the presence or absence of shell calcification in order to characterize gene products involved in the supply of minerals and / or the shell biomineralization process. Results Microarrays were used to identify a repertoire of 302 over-expressed genes during shell calcification. GO terms enrichment was performed to provide a global interpretation of the functions of the over-expressed genes, and revealed that the most over-represented proteins are related to reproductive functions. Our analysis identified 16 gene products encoding proteins involved in mineral supply, and allowed updating of the general model describing uterine ion transporters during eggshell calcification. A list of 57 proteins potentially secreted into the uterine fluid to be active in the mineralization process was also established. They were classified according to their potential functions (biomineralization, proteoglycans, molecular chaperone, antimicrobials and proteases/antiproteases). Conclusions Our study provides detailed descriptions of genes and corresponding proteins over-expressed when the shell is mineralizing. Some of these proteins involved in the supply of minerals and influencing the shell fabric to protect the egg contents are potentially useful biological markers for the genetic improvement of eggshell quality. PMID:24649854

  13. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Rice protein-induced enterocolitis syndrome with transient specific IgE to boiled rice but not to retort-processed rice.

    Science.gov (United States)

    Yasutomi, Motoko; Kosaka, Takuya; Kawakita, Akiko; Hayashi, Hisako; Okazaki, Shintaro; Murai, Hiroki; Miyagawa, Kazuhiko; Mayumi, Mitsufumi; Ohshima, Yusei

    2014-02-01

    Described herein is the case of an 8-month-old girl with atypical food protein-induced enterocolitis syndrome due to rice. She presented with vomiting and poor general activity 2 h after ingestion of boiled rice. Oral food challenge test using high-pressure retort-processed rice was negative, but re-exposure to boiled rice elicited gastrointestinal symptoms. On western blot analysis the patient's serum was found to contain IgE bound to crude protein extracts from rice seed or boiled rice, but not from retort-processed rice. The major protein bands were not detected in the electrophoresed gel of retort-processed rice extracts, suggesting decomposition by high-temperature and high-pressure processing. Oral food challenge for diagnosing rice allergy should be performed with boiled rice to avoid a false negative. Additionally, some patients with rice allergy might be able to ingest retort-processed rice as a substitute for boiled rice. © 2014 The Authors. Pediatrics International © 2014 Japan Pediatric Society.

  15. A sample preparation process for LC-MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody.

    Science.gov (United States)

    Ji, Qin C; Rodila, Ramona; El-Shourbagy, Tawakol A

    2007-03-01

    The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel "denaturing" step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC-MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC-MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r(2)) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.

  16. The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing.

    OpenAIRE

    Ruggero, D; Ciammaruconi, A; Londei, P

    1998-01-01

    The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exist...

  17. 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)

    Belov, O.V.

    2008-01-01

    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

  18. Dietary phosphorus restriction in dialysis patients: potential impact of processed meat, poultry, and fish products as protein sources.

    Science.gov (United States)

    Sherman, Richard A; Mehta, Ojas

    2009-07-01

    Dietary intake of phosphorus is derived largely from protein sources and is a critical determinant of phosphorus balance in patients with chronic kidney disease. Information about the phosphorus content of prepared foods generally is unavailable, but it is believed to contribute significantly to the phosphorus burden of patients with chronic kidney disease. Analysis of dietary components. We measured the phosphorus content of 44 food products, including 30 refrigerated or frozen precooked meat, poultry, and fish items, generally national brands. Measured and reported phosphorus content of foods. Phosphorus by using Association of Analytical Communities official method 984.27; protein by using Association of Analytical Communities official method 990.03. We found that the ratio of phosphorus to protein content in these items ranged from 6.1 to 21.5 mg of phosphorus per 1 g of protein. The mean ratio in the 19 food products with a label listing phosphorus as an additive was 14.6 mg/g compared with 9.0 mg/g in the 11 items without listed phosphorus. The phosphorus content of only 1 precooked food product was available in a widely used dietary database. Results cannot be extrapolated to other products. Manufacturers also may alter the phosphorus content of foods at any time. Protein content was not directly measured for all foods. Better reporting of phosphorus content of foods by manufacturers could result in improved dietary phosphorus control without risk of protein malnutrition.

  19. LIN-61, one of two Caenorhabditis elegans malignant-brain-tumor-repeat-containing proteins, acts with the DRM and NuRD-like protein complexes in vulval development but not in certain other biological processes.

    Science.gov (United States)

    Harrison, Melissa M; Lu, Xiaowei; Horvitz, H Robert

    2007-05-01

    Vulval development in Caenorhabiditis elegans is inhibited by the redundant functions of the synthetic multivulva (synMuv) genes. At least 26 synMuv genes have been identified, many of which appear to act via transcriptional repression. Here we report the molecular identification of the class B synMuv gene lin-61, which encodes a protein composed of four malignant brain tumor (MBT) repeats. MBT repeats, domains of approximately 100 amino acids, have been found in multiple copies in a number of transcriptional repressors, including Polycomb-group proteins. MBT repeats are important for the transcriptional repression mediated by these proteins and in some cases have been shown to bind modified histones. C. elegans contains one other MBT-repeat-containing protein, MBTR-1. We demonstrate that a deletion allele of mbtr-1 does not cause a synMuv phenotype nor does mbtr-1 appear to act redundantly with or in opposition to lin-61. We further show that lin-61 is phenotypically and biochemically distinct from other class B synMuv genes. Our data indicate that while the class B synMuv genes act together to regulate vulval development, lin-61 functions separately from some class B synMuv proteins in other biological processes.

  20. Induction of the unfolded protein response by cigarette smoke is primarily an activating transcription factor 4-C/EBP homologous protein mediated process

    Directory of Open Access Journals (Sweden)

    Geraghty P

    2011-06-01

    Full Text Available Patrick Geraghty, Alison Wallace, Jeanine M D'ArmientoDepartment of Medicine, Divisions of Molecular and Pulmonary Medicine, Columbia University College of Physicians and Surgeons, New York, NY, USAPurpose: Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease (COPD. Recent studies propose a link between endoplasmic reticulum (ER stress and emphysema, demonstrated by increased ER stress markers under smoking conditions. Here, we investigate whether cigarette smoke-induced ER stress is cell specific and correlates with acute and chronic cigarette smoke exposure.Methods: Gene and protein expression changes in human primary lung cell cultures following cigarette smoke extract (CSE exposure were monitored by qPCR and Western blot analysis. Mice and guinea pigs were exposed to cigarette smoke and ER stress markers examined in whole lung homogenates. Inflammatory cells from the bronchoalveolar lavage fluid of 10 days smoke exposed mice were also examined.Results: Cigarette smoke induced a trend increase in the ER stress response through an activating transcription factor 4 (ATF4 mediated induction of C/EBP homologous protein (CHOP in primary small airway epithelial cells. Bronchial epithelial cells and macrophages responded similarly to CSE. Wild-type mice and guinea pigs exposed to acute levels of cigarette smoke exhibited increased levels of CHOP but not at significant levels. However, after long-term chronic cigarette smoke exposure, CHOP expression was reduced. Interestingly, inflammatory cells from smoke exposed mice had a significant increase in CHOP/ATF4 expression.Conclusion: A trend increase in CHOP levels appear in multiple human lung cell types following acute cigarette smoke exposure in vitro. In vivo, inflammatory cells, predominately macrophages, demonstrate significant cigarette smoke-induced ER stress. Early induction of CHOP in cigarette smoke may play a pivotal role in early

  1. The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

  2. Enzymolysis kinetics and activities of ACE inhibitory peptides from wheat germ protein prepared with SFP ultrasound-assisted processing.

    Science.gov (United States)

    Qu, Wenjuan; Ma, Haile; Jia, Junqiang; He, Ronghai; Luo, Lin; Pan, Zhongli

    2012-09-01

    There is a great demand for developing efficient enzymolysis methods in order to increase the enzymolysis efficiencies and activities of angiotensin converting enzyme (ACE) inhibitory peptides from wheat germ protein. The enzymolysis kinetics, ACE inhibitory activity of peptide and conversion rate of protein were studied using sweep frequency and pulsed (SFP) ultrasound-assisted enzymolysis and the results were compared with traditional enzymolysis. The studied factors were enzymolysis time and substrate concentration. By considering the activity of ACE inhibitory peptide and operation cost, the recommended conditions of SFP ultrasound-assisted enzymolysis were enzymolysis time of 120 min and substrate concentration of 24.0 g/L, which gave high conversion rates of protein (60.7%) and ACE inhibitory activity of peptide (65.9%). Compared to traditional enzymolysis, SFP ultrasound-assisted enzymolysis significantly increased the initial reaction rate (V) by 60.0% at substrate concentration of 24.0 g/L, increased the apparent breakdown rate constant (k(A)) by 66.7%, decreased the apparent constant (K(M)) by 6.9%, and raised the conversion rate of protein by 35.5% and ACE inhibitory activity of peptides by 35.6% under the recommended conditions. It has been concluded that SFP ultrasound can remarkably raise the enzymolysis efficiency and activity of ACE inhibitory peptides from wheat germ protein. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Host-Virus Protein Interaction Network Reveals the Involvement of Multiple Host Processes in the Life Cycle of Hepatitis E Virus.

    Science.gov (United States)

    Subramani, Chandru; Nair, Vidya P; Anang, Saumya; Mandal, Sukhen Das; Pareek, Madhu; Kaushik, Nidhi; Srivastava, Akriti; Saha, Sudipto; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

    2018-01-01

    Comprehensive knowledge of host-pathogen interactions is central to understand the life cycle of a pathogen and devise specific therapeutic strategies. Protein-protein interactions (PPIs) are key mediators of host-pathogen interactions. Hepatitis E virus (HEV) is a major cause of viral hepatitis in humans. Recent reports also demonstrate its extrahepatic manifestations in the brain. Toward understanding the molecular details of HEV life cycle, we screened human liver and fetal brain cDNA libraries to identify the host interaction partners of proteins encoded by genotype 1 HEV and constructed the virus-host PPI network. Analysis of the network indicated a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed the presence of multiple host translation regulatory factors in the viral translation/replication complex. Depletion of host translation factors such as eIF4A2, eIF3A, and RACK1 significantly reduced the viral replication, whereas eIF2AK4 depletion had no effect. These findings highlight the ingenuity of the pathogen in manipulating the host machinery to its own benefit, a clear understanding of which is essential for the identification of strategic targets and development of specific antivirals against HEV. IMPORTANCE Hepatitis E virus (HEV) is a pathogen that is transmitted by the fecal-oral route. Owing to the lack of an efficient laboratory model, the life cycle of the virus is poorly understood. During the course of infection, interactions between the viral and host proteins play essential roles, a clear understanding of which is essential to decode the life cycle of the virus. In this study, we identified the direct host interaction partners of all HEV proteins and generated a PPI network. Our functional analysis of the HEV-human PPI network reveals a role of HEV proteins in modulating

  4. 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.

  5. 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

    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 expression...... upon maturation. The protein expression profile of immature and mature DCs, derived from CD14+ peripheral blood monocytes was investigated using two pH ranges (pH 4-7 and 6-9) (n = 4). Ninety one differentially expressed spots (p...

  6. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein

    OpenAIRE

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-01-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackieviru...

  7. Influences of process and formulation parameters on powder flow properties and immunogenicity of spray dried polymer particles entrapping recombinant pneumococcal surface protein A.

    Science.gov (United States)

    Anish, Chakkumkal; Upadhyay, Arun K; Sehgal, Devinder; Panda, Amulya Kumar

    2014-05-15

    Particle size, antigen load and its release characteristic are the three the main attributes of polymer particles based vaccine delivery systems. The present studies focus on the formulation of spray dried polylactide microparticles entrapping pneumococcal surface protein A (PspA). Influence of process variables during polymer particle formation were optimized by using half-factorial design. Feed rate and atomization pressure during spray drying were found to be the most important parameters for achieving uniform size particles. Spray drying of preformed particles from different stages of solvent evaporation method resulted in formation of particle having different porosity and protein release profile. Presence of polyvinyl alcohol in the external aqueous phase not only contributed towards regulating the size of particles but also influenced the burst release of protein from particles. Polymer particles entrapping PspA elicited robust IgG responses both in mice and in rats. Antigen load in microparticles correlated with the antibody titer indicating the maintenance of protein integrity during particle formation using spray drying. Both, process engineering and formulation parameters during spray drying influenced the particles in terms of size, load and antigen release characteristics. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins.

    Science.gov (United States)

    Buyel, Johannes F; Opdensteinen, Patrick; Fischer, Rainer

    2015-04-01

    Downstream processing (DSP) is a major cost factor during the production of biopharmaceutical proteins. Clarification can account for ∼40% of these costs, especially when a large amount of dispersed particulate material is generated, such as during the extraction of intracellular proteins from plants. Filter capacity can be increased (and DSP costs reduced) by using flocculants. Here we show that cellulose-based filter aids can enhance the positive effect of flocculants by improving depth filter capacity even further. A design-of-experiments (DoE) approach was used to identify the optimal size and concentration of filter aids, at different values of pH and conductivity, for the clarification of tobacco leaf extracts during the production of a monoclonal antibody and a fluorescent protein. Filter aids ∼28 or ∼100 μm in length at concentrations of ∼10 and ∼5 g L(-1) respectively were most efficient in combination with a strong cationic flocculant, but were ineffective without the flocculant. The filter aids increased depth filter capacity by 35-fold compared to an additive-free extract reaching ∼1000 L m(-2) without affecting the target proteins. Thus, filter aids can be used to reduce production costs of plant-derived biopharmaceuticals while the DoE approach enabled the identification of robust process conditions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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

    2015-12-05

    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 (Ppeas had lower (Ppea-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. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Comparative genomic analysis of a neurotoxigenic Clostridium species using partial genome sequence: Phylogenetic analysis of a few conserved proteins involved in cellular processes and metabolism.

    Science.gov (United States)

    Alam, Syed Imteyaz; Dixit, Aparna; Tomar, Arvind; Singh, Lokendra

    2010-04-01

    Clostridial organisms produce neurotoxins, which are generally regarded as the most potent toxic substances of biological origin and potential biological warfare agents. Clostridium tetani produces tetanus neurotoxin and is responsible for the fatal tetanus disease. In spite of the extensive immunization regimen, the disease is an important cause of death especially among neonates. Strains of C. tetani have not been genetically characterized except the complete genome sequencing of strain E88. The present study reports the genetic makeup and phylogenetic affiliations of an environmental strain of this bacterium with respect to C. tetani E88 and other clostridia. A shot gun library was constructed from the genomic DNA of C. tetani drde, isolated from decaying fish sample. Unique clones were sequenced and sequences compared with its closest relative C. tetani E88. A total of 275 clones were obtained and 32,457 bases of non-redundant sequence were generated. A total of 150 base changes were observed over the entire length of sequence obtained, including, additions, deletions and base substitutions. Of the total 120 ORFs detected, 48 exhibited closest similarity to E88 proteins of which three are hypothetical proteins. Eight of the ORFs exhibited similarity with hypothetical proteins from other organisms and 10 aligned with other proteins from unrelated organisms. There is an overall conservation of protein sequences among the two strains of C. tetani and. Selected ORFs involved in cellular processes and metabolism were subjected to phylogenetic analysis. Copyright 2009 Elsevier Ltd. All rights reserved.

  11. Enrichment and purification of casein glycomacropeptide from whey protein isolate using supercritical carbon dioxide processing and membrane filtration

    Science.gov (United States)

    Whey protein concentrates (WPC) and isolates (WPI), which are dried, concentrated forms of cheese whey, are comprised mainly of beta–lactoglobulin (beta-LG), a–lactalbumin (a-LA), and glycomacropeptide (GLY), and are added to foods to boost their nutritional and functional properties. In previous st...

  12. 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 ...

  13. Modulation of proteolytic polyprotein processing by coxsackievirus mutants resistant to inhibitors targeting phosphatidylinositol-4-kinase IIIβ or oxysterol binding protein

    NARCIS (Netherlands)

    Lyoo, Heyrhyoung; Dorobantu, Cristina M; van der Schaar, Hilde M; van Kuppeveld, Frank J M

    2017-01-01

    Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIβ (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were

  14. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification.

    Science.gov (United States)

    Zheng, Ji; Wang, Lu; Twarowska, Barbara; Laino, Sarah; Sparks, Colleen; Smith, Timothy; Russell, Reb; Wang, Michelle

    2015-01-01

    This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers.

  16. A novel aqueous two phase assisted platform for efficient removal of process related impurities associated with E. coli based biotherapeutic protein products.

    Science.gov (United States)

    Bhambure, Rahul; Sharma, Rohit; Gupta, Darpan; Rathore, Anurag S

    2013-09-13

    This article presents a variant of aqueous two phase system (ATPS) as a tool for selective removal of process related impurities associated with Escherichia coli, namely host cell proteins and nucleic acids. Granulocyte colony stimulating factor (GCSF) expressed in E. coli has been selected as a model protein for the study. While achieving effective removal of host cell impurities as per the regulatory requirement for recombinant therapeutics, high product recovery has been achieved by adopting a novel strategy involving resolubilization of interfacial GCSF precipitate. This has been done such that the structural and biological activity of the product is retained. Exhaustive analysis of structural as well as functional integrity of resolubilized GCSF has been carried out using advanced analytical and in vitro bioassay tools. Product recovery of 99.5% has been achieved with the concentration of host cell proteins less than 100ppm and of nucleic acids below 10ng/ml. We think that the proposed platform can enable use of ATPS as a more economical alternative to process chromatography in industrial biopharmaceutical manufacturing processes. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Process Analytical Approach towards Quality Controlled Process Automation for the Downstream of Protein Mixtures by Inline Concentration Measurements Based on Ultraviolet/Visible Light (UV/VIS Spectral Analysis

    Directory of Open Access Journals (Sweden)

    Steffen Zobel-Roos

    2017-12-01

    Full Text Available Downstream of pharmaceutical proteins, such as monoclonal antibodies, is mainly done by chromatography, where concentration determination of coeluting components presents a major problem. Inline concentration measurements (ICM by Ultraviolet/Visible light (UV/VIS-spectral data analysis provide a label-free and noninvasive approach to significantly speed up the analysis and process time. Here, two different approaches are presented. For a test mixture of three proteins, a fast and easily calibrated method based on the non-negative least-squares algorithm is shown, which reduces the calibration effort compared to a partial least-squares approach. The accuracy of ICM for analytical separations of three proteins on an ion exchange column is over 99%, compared to less than 85% for classical peak area evaluation. The power of the partial least squares algorithm (PLS is shown by measuring the concentrations of Immunoglobulin G (IgG monomer and dimer under a worst-case scenario of completely overlapping peaks. Here, the faster SIMPLS algorithm is used in comparison to the nonlinear iterative partial least squares (NIPALS algorithm. Both approaches provide concentrations as well as purities in real-time, enabling live-pooling decisions based on product quality. This is one important step towards advanced process automation of chromatographic processes. Analysis time is less than 100 ms and only one program is used for all the necessary communications and calculations.

  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 larvae gut soluble proteases, yielding derivative proteins essentially of about 62 and 33 kDa. The gut-soluble proteases could contain trypsin-like enzymes implicated in Vip3Aa16 activation since the proteolysis was inhibited using specific protease inhibitors. Additionally, we showed that the histopathological effect of Vip3Aa16 on T. absoluta larva midguts consisted on a microvillus damage and an epithelial cell rupture.

  19. 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.

  20. Crystallographic studies with xenon and nitrous oxide provide evidence for protein-dependent processes in the mechanisms of general anesthesia.

    Science.gov (United States)

    Abraini, Jacques H; Marassio, Guillaume; David, Helene N; Vallone, Beatrice; Prangé, Thierry; Colloc'h, Nathalie

    2014-11-01

    The mechanisms by which general anesthetics, including xenon and nitrous oxide, act are only beginning to be discovered. However, structural approaches revealed weak but specific protein-gas interactions. To improve knowledge, we performed x-ray crystallography studies under xenon and nitrous oxide pressure in a series of 10 binding sites within four proteins. Whatever the pressure, we show (1) hydrophobicity of the gas binding sites has a screening effect on xenon and nitrous oxide binding, with a threshold value of 83% beyond which and below which xenon and nitrous oxide, respectively, binds to their sites preferentially compared to each other; (2) xenon and nitrous oxide occupancies are significantly correlated respectively to the product and the ratio of hydrophobicity by volume, indicating that hydrophobicity and volume are binding parameters that complement and oppose each other's effects; and (3) the ratio of occupancy of xenon to nitrous oxide is significantly correlated to hydrophobicity of their binding sites. These data demonstrate that xenon and nitrous oxide obey different binding mechanisms, a finding that argues against all unitary hypotheses of narcosis and anesthesia, and indicate that the Meyer-Overton rule of a high correlation between anesthetic potency and solubility in lipids of general anesthetics is often overinterpreted. This study provides evidence that the mechanisms of gas binding to proteins and therefore of general anesthesia should be considered as the result of a fully reversible interaction between a drug ligand and a receptor as this occurs in classical pharmacology.

  1. Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system.

    Science.gov (United States)

    Sun, He; Zhou, Ni; Wang, Hai; Huang, Dafang; Lang, Zhihong

    2017-01-01

    In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV) causes the co-translational "cleavage" of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing. LP4/2A is a hybrid linker peptide that contains the first nine amino acids of LP4 and 20 amino acids of 2A. The three linkers have been used as a suitable technique to link the expression of genes in some transgenic plants, but to date the cleavage efficiency of three linkers have not been comprehensively demonstrated in the same transformation system, especially in the staple crop. To verify the functions of 2A, LP4, and LP4/2A linker peptides in transgenic maize, six fusion protein vectors that each encoded a single open reading frame (ORF) incorporating two report genes, Green Fluorescent Protein (GFP) and β-glucuronidase (GUS), separated by 2A (or modified 2A), LP4 or LP4/2A were assembled to compare the cleavage efficiency of the three linkers in a maize transient expression system. The results demonstrated the more protein production and higher cleavage splicing efficiency with the polyprotein construct linked by the LP4/2A peptide than those of the polyprotein constructs linked by 2A or LP4 alone. Seven other fusion proteins that each encoded a single ORF incorporating two different genes GFP and Red Fluorecent Protein (RFP) with different signal peptides were assembled to study the subcellular localization of genes linked by LP4/2A. The subcellular localization experiments suggested that both types of signal peptide, co-translational and post-translational, could lead their proteins to the target localization in maize

  2. Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system.

    Directory of Open Access Journals (Sweden)

    He Sun

    Full Text Available In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV causes the co-translational "cleavage" of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing. LP4/2A is a hybrid linker peptide that contains the first nine amino acids of LP4 and 20 amino acids of 2A. The three linkers have been used as a suitable technique to link the expression of genes in some transgenic plants, but to date the cleavage efficiency of three linkers have not been comprehensively demonstrated in the same transformation system, especially in the staple crop. To verify the functions of 2A, LP4, and LP4/2A linker peptides in transgenic maize, six fusion protein vectors that each encoded a single open reading frame (ORF incorporating two report genes, Green Fluorescent Protein (GFP and β-glucuronidase (GUS, separated by 2A (or modified 2A, LP4 or LP4/2A were assembled to compare the cleavage efficiency of the three linkers in a maize transient expression system. The results demonstrated the more protein production and higher cleavage splicing efficiency with the polyprotein construct linked by the LP4/2A peptide than those of the polyprotein constructs linked by 2A or LP4 alone. Seven other fusion proteins that each encoded a single ORF incorporating two different genes GFP and Red Fluorecent Protein (RFP with different signal peptides were assembled to study the subcellular localization of genes linked by LP4/2A. The subcellular localization experiments suggested that both types of signal peptide, co-translational and post-translational, could lead their proteins to the target

  3. 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.

  4. Optimal model-based design of the twin-column CaptureSMB process improves capacity utilization and productivity in protein A affinity capture.

    Science.gov (United States)

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

    2016-01-01

    Multi-column chromatographic processes have recently been developed for protein A affinity chromatography to efficiently capture monoclonal antibodies from cell culture supernatant. In this work, the novel twin-column CaptureSMB process was compared to a batch capture process with dual loading flow rate to identify performance gains. As a case study, the isolation of a monoclonal antibody with the Amsphere JWT-203 protein A resin was investigated. Using model based optimization, both processes were optimized and compared over a wide range of operating conditions. A trade-off between productivity and capacity utilization was found, and the resulting pareto-curves showed that CaptureSMB dominates batch, except at very low productivity values. With a feed titer of 1.2 mg mL(-1) , CaptureSMB could reach a productivity of up to 19.5 mg mL(-1) h(-1) experimentally, while maintaining relatively high capacity utilization of 63.8%. On the other hand, at maximum capacity utilization of 95.5%, a productivity of 10.2 mg mL(-1) h(-1) could be reached. This corresponds to a performance improvement with respect batch operation of about 25% in capacity utilization and 40% in productivity, for given yield and purity. CaptureSMB therefore offers a greatly increased performance over batch capture. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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

    2015-09-02

    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

  6. 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.

  7. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling.

    Science.gov (United States)

    Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

    2013-12-06

    An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from N(pro) fusion pep6His and N(pro) fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes - one using urea for IB dissolution, the other one using NaOH for IB dissolution - and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Recent research in flaxseed (oil seed) on molecular structure and metabolic characteristics of protein, heat processing-induced effect and nutrition with advanced synchrotron-based molecular techniques.

    Science.gov (United States)

    Doiron, Kevin J; Yu, Peiqiang

    2017-01-02

    Advanced synchrotron radiation-based infrared microspectroscopy is able to reveal feed and food structure feature at cellular and molecular levels and simultaneously provides composition, structure, environment, and chemistry within intact tissue. However, to date, this advanced synchrotron-based technique is still seldom known to food and feed scientists. This article aims to provide detailed background for flaxseed (oil seed) protein research and then review recent progress and development in flaxseed research in ruminant nutrition in the areas of (1) dietary inclusion of flaxseed in rations; (2) heat processing effect; (3) assessing dietary protein; (4) synchrotron-based Fourier transform infrared microspectroscopy as a tool of nutritive evaluation within cellular and subcellular dimensions; (5) recent synchrotron applications in flaxseed research on a molecular basis. The information described in this paper gives better insight in flaxseed research progress and update.

  10. Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging

    DEFF Research Database (Denmark)

    Karring, Henrik; Runager, Kasper; Valnickova, Zuzana

    2010-01-01

    Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging....... TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 mug TGFBIp per mg wet tissue. Two-dimentional (2-D......) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (corneas (>6 years). Matrix...

  11. 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

    AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

  12. 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

    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 proteinase inhibitor leupeptin partially restored the suppressed synthesis, indicating that the majority of the wrongly processed enzymes, probably because of conformational instability, become degraded soon after synthesis rather than being transported to the microvillar membrane....

  13. TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

    Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

  14. Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2).

    Science.gov (United States)

    Frykman, Susanne; Inoue, Mitsuhiro; Ikeda, Atsushi; Teranishi, Yasuhiro; Kihara, Takahiro; Lundgren, Jolanta L; Yamamoto, Natsuko G; Bogdanovic, Nenad; Winblad, Bengt; Schedin-Weiss, Sophia; Tjernberg, Lars O

    2017-01-29

    The toxic amyloid β-peptide (Aβ) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase (to yield APP-C-terminal fragment β (APP-CTFβ) and soluble APPβ (sAPPβ)) and γ-secretase (to yield Aβ). We reasoned that proteins that associate with γ-secretase are likely to regulate Aβ production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aβ levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aβ, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aβ levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by β-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

    Science.gov (United States)

    Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

    2017-09-15

    Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

  16. 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)

    Kitagawa, Yukiko; Kameoka, Masanori; Shoji-Kawata, Sanae; Iwabu, Yukie; Mizuta, Hiroyuki; Tokunaga, Kenzo; Fujino, Masato; Natori, Yukikazu; Yura, Yoshiaki; Ikuta, Kazuyoshi

    2008-01-01

    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

  17. Penicillium antifungal protein (PAF) is involved in the apoptotic and autophagic processes of the producer Penicillium chrysogenum.

    Science.gov (United States)

    Kovács, Barbara; Hegedűs, Nikoletta; Bálint, Mihály; Szabó, Zsuzsa; Emri, Tamás; Kiss, Gréta; Antal, Miklós; Pócsi, István; Leiter, Eva

    2014-09-01

    PAF, which is produced by the filamentous fungus Pencicillium chrysogenum, is a small antifungal protein, triggering ROS-mediated apoptotic cell death in Aspergillus nidulans. In this work, we provide information on the function of PAF in the host P. chrysogenum considering that carbon-starving cultures of the Δpaf mutant strain showed significantly reduced apoptosis rates in comparison to the wild-type (wt) strain. Moreover, the addition of PAF to the Δpaf strain resulted in a twofold increase in the apoptosis rate. PAF was also involved in the regulation of the autophagy machinery of this fungus, since several Saccharomyces cerevisiae autophagy-related ortholog genes, e.g. those of atg7, atg22 and tipA, were repressed in the deletion strain. This phenomenon was accompanied by the absence of autophagosomes in the Δpaf strain, even in old hyphae.

  18. 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 Shivaya; 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

  19. Mapping the brain pathways of traumatic memory: inactivation of protein kinase M zeta in different brain regions disrupts traumatic memory processes and attenuates traumatic stress responses in rats.

    Science.gov (United States)

    Cohen, Hagit; Kozlovsky, Nitsan; Matar, Michael A; Kaplan, Zeev; Zohar, Joseph

    2010-04-01

    Protein kinase M zeta (PKMzeta), a constitutively active isoform of protein kinase C, has been implicated in protein synthesis-dependent maintenance of long-term potentiation and memory storage in the brain. Recent studies reported that local application of ZIP, a membrane-permeant PKMzeta inhibitor, into the insular cortex (IC) of behaving rats abolished long-term memory of taste associations. This study assessed the long-term effects of local applications of ZIP microinjected immediately (1 h) or 10 days after predator scent stress exposure, in a controlled prospectively designed animal model for PTSD. Four brain structures known to be involved in memory processes and in anxiety were investigated: lateral ventricle (LV), dorsal hippocampus (DH), basolateral amygdala and IC. The outcome measures included behavior in an elevated plus maze and acoustic startle response 7 days after microinjection, and freezing behavior upon exposure to trauma-related cue 8 days after microinjection. Previously acquired/encoded memories associated with the IC were also assessed. Inactivation of PKMzeta in the LV or DH within 1h of exposure effectively reduced PTSD-like behavioral disruption and trauma cue response 8 days later. Inactivation of PKMzeta 10 days after exposure had equivalent effects only when administered in the IC. The effect was demonstrated to be specific for trauma memories, whereas previously acquired data were unaffected by the procedure. Predator scent related memories are located in different brain areas at different times beginning with an initial hippocampus-dependent consolidation process, and are eventually stored in the IC. These bring the IC to the forefront as a potential region of significance in processes related to traumatic stress-induced disorders. 2010 Elsevier B.V. and ECNP. All rights reserved.

  20. Modeling and computations of the intramolecular electron transfer process in the two-heme protein cytochrome c4

    DEFF Research Database (Denmark)

    Natzmutdinov, Renat R.; Bronshtein, Michael D.; Zinkicheva, Tamara T.

    2012-01-01

    ligands in both low- and high-spin states by structure-optimized DFT. The computations enable estimating the intramolecular reorganization energy of the ET process for different combinations of low- and high-spin heme couples. Environmental reorganization free energies, work terms (‘‘gating’’) and driving...... performed computational modeling of the intramolecular ET process by a combination of density functional theory (DFT) and quantum mechanical charge transfer theory to disclose reasons for this difference. We first address the electronic structures of the model heme core with histidine and methionine axial...

  1. Seventeen copies of the human 37 kDa laminin receptor precursor/p40 ribosome-associated protein gene are processed pseudogenes arisen from retropositional events

    DEFF Research Database (Denmark)

    Jackers, P; Clausse, N; Fernandez, M

    1996-01-01

    A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed...... genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons...

  2. Varicellovirus UL49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

    NARCIS (Netherlands)

    Koppers-Lalic, D.; Verweij, M.C.; Lipinska, A.D.; Wang, Y.; Quinten, E.; Reits, E.A.; Koch, J.; Loch, S.; Rezende, M.M.; Daus, F.J.; Bienkowska-Szewczyk, K.; Osterrieder, N.; Mettenleiter, T.C.; Heemskerk, M.H.M.; Tampe, R.; Neefjes, J.J.; Chowdhury, S.I.; Ressing, M.E.; Rijsewijk, F.A.M.; Wiertz, E.J.H.J.

    2008-01-01

    Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing

  3. Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

    NARCIS (Netherlands)

    Koppers-Lalic, Danijela; Verweij, Marieke C.; Lipińska, Andrea D.; Wang, Ying; Quinten, Edwin; Reits, Eric A.; Koch, Joachim; Loch, Sandra; Rezende, Marisa Marcondes; Daus, Franz; Bieńkowska-Szewczyk, Krystyna; Osterrieder, Nikolaus; Mettenleiter, Thomas C.; Heemskerk, Mirjam H. M.; Tampé, Robert; Neefjes, Jacques J.; Chowdhury, Shafiqul I.; Ressing, Maaike E.; Rijsewijk, Frans A. M.; Wiertz, Emmanuel J. H. J.

    2008-01-01

    Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing

  4. 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.

    2015-01-01

    : The 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...

  5. 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: 0.687, year: 2015

  6. Cloning, sequencing and expression in the dog of the main amyloid precursor protein isoforms and some of the enzymes related with their processing.

    Science.gov (United States)

    Sarasa, L; Gallego, C; Monleón, I; Olvera, A; Canudas, J; Montañés, M; Pesini, P; Sarasa, M

    2010-12-29

    Alzheimer's disease (AD) is characterized by neuronal loss and the presence of both neurofibrillary tangles and senile plaques in the brain. These plaques arise from the deposition of beta-amyloid (Aβ) peptides (38-43 amino acids), which are generated from enzymatic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In the present work, we cloned the principal APP isoforms as well as some enzymes that have been implicated in their amyloidogenic and non-amyloidogenic processing in dogs. Additionally, the main proteases implicated in the degradation of Aβ were also studied. We also investigated the level of expression of these APP isoforms and enzymes in different brain regions and in peripheral tissues. Our data demonstrate that these canine proteins are highly homologous to their human counterparts. In addition, the expression pattern of these proteins in dogs is consistent with previous data reported in human beings. Thus, dogs may be a natural model to study the biology of AD and could also serve as an animal model for Aβ-targeted drugs against this devastating disease. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. 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.

  8. Chemical modification of protein a chromatography ligands with polyethylene glycol. II: Effects on resin robustness and process selectivity.

    Science.gov (United States)

    Weinberg, Justin; Zhang, Shaojie; Kirkby, Allison; Shachar, Enosh; Carta, Giorgio; Przybycien, Todd

    2018-04-20

    We have proposed chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) as a strategy to increase the resin selectivity and robustness by providing the ligand with a steric repulsion barrier against non-specific binding. Here, we report on robustness and selectivity benefits for Repligen CaptivA PriMAB resin with ligands modified with 5.2 kDa and 21.5 kDa PEG chains, respectively. PEGylation of ProA ligands allowed the resin to retain a higher percentage of static binding capacity relative to the unmodified resin upon digestion with chymotrypsin, a representative serine protease. The level of protection against digestion was independent of the PEG molecular weight or modification extent for the PEGylation chemistry used. Additionally, PEGylation of the ligands was found to decrease the level of non-specific binding of fluorescently labeled bovine serum albumin (BSA) aggregates to the surface of the resin particles as visualized via confocal laser scanning microscopy (CLSM). The level of aggregate binding decreased as the PEG molecular weight increased, but increasing the extent of modification with 5.2 kDa PEG chains had no effect. Further examination of resin particles via CLSM confirmed that the PEG chains on the modified ligands were capable of blocking the "hitchhiking" association of BSA, a mock contaminant, to an adsorbed mAb that is prone to BSA binding. Ligands modified with 21.5 kDa PEG chains were effective at blocking the association, while ligands modified with 5.2 kDa PEG chains were not. Finally, ligands with 21.5 kDa PEG chains increased the selectivity of the resin against host cell proteins (HCPs) produced by Chinese Hamster Ovary (CHO) cells by up to 37% during purification of a monoclonal antibody (mAb) from harvested cell culture fluid (HCCF) using a standard ProA chromatography protocol. The combined work suggests that PEGylating ProA chromatography media is a viable pathway for

  9. Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive beta-1,4-endoglucanase from Cellulomonas flavigena.

    Science.gov (United States)

    Mejia-Castillo, Teresa; Hidalgo-Lara, Maria Eugenia; Brieba, Luis G; Ortega-Lopez, Jaime

    2008-04-01

    A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60 degrees C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.

  10. Abelson interactor-1 (ABI-1) interacts with MRL adaptor protein MIG-10 and is required in guided cell migrations and process outgrowth in C.elegans

    Science.gov (United States)

    McShea, Molly A.; Schmidt, Kristopher L.; Dubuke, Michelle L.; Baldiga, Christina E.; Sullender, Meagan E.; Reis, Andrea L.; Zhang, Subaiou; O'Toole, Sean M.; Jeffers, Mary C.; Warden, Rachel M.; Kenney, Allison H.; Gosselin, Jennifer; Kuhlwein, Mark; Hashmi, Sana K.; Stringham, Eve G.; Ryder, Elizabeth F.

    2012-01-01

    Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or ‘canals’ of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-mmunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large

  11. 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.

  12. Post-translational modified proteins are biomarkers of autoimmune-processes: NETosis and the inflammatory-autoimmunity connection.

    Science.gov (United States)

    Bruschi, Maurizio; Petretto, Andrea; Bertelli, Roberta; Galetti, Maricla; Bonanni, Alice; Pratesi, Federico; Migliorini, Paola; Candiano, Giovanni; Vaglio, Augusto; Ghiggeri, Gian Marco

    2017-01-01

    Basic research is showing new mechanisms involved in early immune responses and Neutrophil Extracellular Trap (NET) formation (or NETosis) is of key importance as first line defense against bacteria, virus and protozoa. Enzymatic modification of arginine in citrulline in histones is the prerequisite of NETosis being it necessary for decondensation and extrusion of DNA from cells; it is conceivable that other post translational modifications may occur during this event. There is consensus in considering that post translational modified proteins may elicit an autoimmune response that leads to the formation of autoantibodies. Several autoimmune diseases seem to share these pathogenic mechanisms, in particular Rheumatoid arthritis, Systemic Lupus Erythematosus, Small Vessel Vasculitis and Anti-Phospholipid Syndrome, which are all characterized by high levels of circulating autoantibodies. Autoimmunity has, however, different targets and elicits different clinical responses. It seems reasonable to hypothesize that although NETosis is common to all the conditions above, NET components are different and potentially responsible for different autoimmune responses. On the other hand also showing whether circulating NET remnants are present as free structures in blood/biological fluids and determine their levels is relevant to autoimmunity. This review is intended to discuss the rationale for utilizing new discoveries that could be of rapid clinical application and lead to the development of early biomarkers of autoimmunity to predict and treat otherwise serious conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. The effect of the cytoplasmic tail of influenza C virus CM2 protein on its biochemical properties and intracellular processing.

    Science.gov (United States)

    Shimotai, Yoshitaka; Goto, Takanari; Matsuzaki, Yoko; Muraki, Yasushi; Sugawara, Kanetsu; Hongo, Seiji

    2015-09-01

    CM2 is an integral membrane protein encoded by the influenza C virus M gene. To examine the effects of the cytoplasmic tail of CM2 on its biochemical properties, deletion and substitution mutations were introduced into CM2 cytoplasmic tail at residues 47-115, and the expressed CM2 mutants were investigated. Although the cytoplasmic tail is not essential for the oligomerization of CM2, it may affect the degree of oligomerization. The residues 47-48, 67-69, 73-90 and 113-115 were all required for the proper expression of CM2. Pulse-chase experiments suggest that residues 47-48, 67-69, 73-75 and 79-87 stabilize CM2, thereby affecting CM2 expression. The C-terminal region at residues 61-115 is not essential for CM2 transport to the cell surface, and a 14-amino-acid, but not an 11-amino-acid, cytoplasmic tail is sufficient for the cell surface expression of CM2. These results suggest that either certain amino acid sequences or the length of the CM2 cytoplasmic tail are necessary for the proper conformational maturation, stability, expression level and intracellular transport of CM2.

  14. Behavior of leucine-rich repeat-containing G-protein coupled receptor 5-expressing cells in the reprogramming process

    Directory of Open Access Journals (Sweden)

    Yuko Arioka

    2017-04-01

    Full Text Available It remains unclear what cells are proper for the generation of induced pluripotent stem cells (iPSCs. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5 is well known as a tissue stem cell and progenitor marker, both of which are reported to be sensitive to reprogramming. In the present study, we examined the reprogramming behavior of Lgr5-expressing cells (Lgr5+ cells. First, we compared reprogramming behavior using mouse Lgr5+ and Lgr5 negative (Lgr5− hair follicles (HFs. The number of alkaline phosphatase staining-positive cells was lesser in a well of Lgr5+ HFs than in Lgr5− HFs; however, the ratio of Nanog+ SSEA1+ cells in the cell mixture derived from Lgr5+ HFs was much higher than that from Lgr5− HFs. Lgr5+ cells could be induced from mouse embryonic fibroblasts (MEFs after transduction with Yamanaka factors. As shown in HFs, the progeny of Lgr5+ cells arising from MEFs highly converted into Nanog+ cells and did not form Nanog− colonies. The progeny represented the status of the late reprogramming phase to a higher degree than the nonprogeny. We also confirmed this using human Lg5+ cells. Our findings suggest that the use of Lgr5+ cells will minimize sorting efforts for obtaining superior iPSCs.

  15. Impact of Processing on the Protein Quality of Pinto Bean (Phaseolus vulgaris) and Buckwheat (Fagopyrum esculentum Moench) Flours and Blends, As Determined by in Vitro and in Vivo Methodologies.

    Science.gov (United States)

    Nosworthy, Matthew G; Franczyk, Adam; Zimoch-Korzycka, Anna; Appah, Paulyn; Utioh, Alphonsus; Neufeld, Jason; House, James D

    2017-05-17

    Blending of protein sources can increase protein quality by compensating for limiting amino acids present in individual sources, whereas processing grain flours by extrusion or baking can also alter protein quality. To determine the effect of baking and extrusion on the protein quality of blended flours from buckwheat and pinto beans, a rodent bioassay was performed and compared to an in vitro method of protein quality determination. Overall, extruded products had higher protein efficiency ratio values, increased digestibility, and greater protein digestibility corrected amino acid score (PDCAAS) values than baked products, with the extruded buckwheat/pinto blend having the greatest PDCAAS value of the experimental diets investigated. A correlation was found between both digestibility and PDCAAS values generated from in vitro and in vivo methods. The use of in vitro digestibility analysis should be investigated as a potential replacement for the current rodent assay for nutrient content claim purposes.

  16. A three-prong strategy to develop functional food using protein isolates recovered from chicken processing by-products with isoelectric solubilization/precipitation.

    Science.gov (United States)

    Tahergorabi, Reza; Sivanandan, Litha; Beamer, Sarah K; Matak, Kristen E; Jaczynski, Jacek

    2012-09-01

    Skin-on bone-in chicken drumsticks were processed with isoelectric solubilization/precipitation to recover muscle proteins. The drumsticks were used as a model for dark chicken meat processing by-products. The main objective of this study was conversion of dark chicken meat processing by-products to restructured functional food product. An attempt was made to develop functional food product that would resemble respective product made from boneless skinless chicken breast meat. A three-prong strategy to address diet-driven cardiovascular disease (CVD)with a functional food was used in this study. The strategy included addition of three ingredients with well-documented cardiovascular benefits: (i) ω-3 polyunsaturated fatty acid-rich oil (flaxseed-algae, 9:1); (ii) soluble fiber; and (iii) salt substitute. Titanium dioxide, potato starch, polyphosphate, and transglutaminase were also added. The batters were formulated and cooked resulting in heat-set gels. Color (L*a*b*), texture (torsion test, Kramer shear test, and texture profile analysis), thermal denaturation (differential scanning calorimetry), and gelation (dynamic rheology) of chicken drumstick gels and chicken breast gels were determined and compared. Chicken drumstick gels generally had comparable color and texture properties to the gels made from chicken breast meat. The endothermic transition (thermal denaturation) of myosin was more pronounced and gelation properties were better for the drumstick gels. This study demonstrated a feasibility to develop functional food made of muscle proteins recovered with isoelectric solubilization/precipitation from low-value dark chicken meat processing by-products. The functional food developed in this study was enriched with CVD-beneficial nutrients and had comparable instrumental quality attributes to respective products made of chicken breast meat. Although the results of this study point towards the potential for a novel, marketable functional food product, sensory

  17. Effect of crude protein concentration and dietary electrolyte balance on litter quality, foot pad dermatitis, growth performance and processing yields in two medium heavy turkey hybrids.

    Science.gov (United States)

    Veldkamp, T; Hocking, P M; Vinco, L J

    2017-10-01

    1. An experiment was conducted to investigate the effect of crude protein (CP) concentration and dietary electrolyte balance (DEB) on growth performance, processing yields, litter quality and foot pad dermatitis (FPD) in male turkeys from two commercial hybrids. Soya bean meal was replaced by vegetable protein sources selected for lower K concentrations to lower DEB in order to improve litter quality and subsequent quality of foot pads. 2. Effects of CP on litter friability and wetness were not consistent during the production period. FPD in turkeys fed on diets with low CP was significantly lower than FPD in turkeys fed on diets with high CP until 84 d. Growth performance was adversely affected at low CP. Processing yields were not affected by CP. 3. Litter was significantly dryer in pens of turkeys fed on diets with low DEB than in pens of turkeys fed on diets with high DEB. FPD in turkeys fed on diets with low DEB was significantly lower than in turkeys fed on diets with high DEB. Growth performance and processing yields were adversely affected at low DEB. 4. FPD in turkey hybrid A was higher than in turkey hybrid B at 28 d of age. Thereafter, no differences in FPD between turkey hybrids were observed. Growth performance and processing yields were not affected by turkey hybrid. 5. Overall, a significant interaction effect of CP × DEB was observed for FCR: in turkeys fed on the high DEB treatment, FCR of turkeys fed on the high CP diets was lower than FCR of turkeys fed on the low CP (LCP) diets whereas on the low DEB treatment, FCR was not affected by CP treatment. 6. It was concluded that litter quality can be improved and FPD may be decreased in turkeys fed on diets containing lower CP and DEB levels.

  18. Ezrin/Radixin/Moesin Are Required for the Purinergic P2X7 Receptor (P2X7R)-dependent Processing of the Amyloid Precursor Protein*

    Science.gov (United States)

    Darmellah, Amaria; Rayah, Amel; Auger, Rodolphe; Cuif, Marie-Hélène; Prigent, Magali; Arpin, Monique; Alcover, Andres; Delarasse, Cécile; Kanellopoulos, Jean M.

    2012-01-01

    The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP. PMID:22891241

  19. Ezrin/radixin/moesin are required for the purinergic P2X7 receptor (P2X7R)-dependent processing of the amyloid precursor protein.

    Science.gov (United States)

    Darmellah, Amaria; Rayah, Amel; Auger, Rodolphe; Cuif, Marie-Hélène; Prigent, Magali; Arpin, Monique; Alcover, Andres; Delarasse, Cécile; Kanellopoulos, Jean M

    2012-10-05

    The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.

  20. 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.

  1. Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences.

    Science.gov (United States)

    Carvalho, Rimenys Junior; Cabrera-Crespo, Joaquin; Tanizaki, Martha Massako; Gonçalves, Viviane Maimoni

    2012-05-01

    Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

  2. 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)

  3. 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

    Incubation of Fe(III)myoglobin (Fe(III)Mb) with H2O2 in the presence of bovine serum albumin (BSA) has been shown previously to give albumin-derived radicals as a result of radical transfer from myoglobin to BSA. In this study the occurrence of similar processes with peroxidases has been investig......Incubation of Fe(III)myoglobin (Fe(III)Mb) with H2O2 in the presence of bovine serum albumin (BSA) has been shown previously to give albumin-derived radicals as a result of radical transfer from myoglobin to BSA. In this study the occurrence of similar processes with peroxidases has been...... investigated using horseradish peroxidase (HRP)/H2O2, in the presence and absence of added tyrosine. Incubation of HRP with H2O2 and bovine or human serum albumins, in the presence and absence of tyrosine, gave long-lived albumin-derived radicals as detected by EPR spectroscopy. Evidence has been obtained...... concentrations were observed after limited digestion, although this effect was less marked with the HRP/H2O2/BSA system than with Fe(III)Mb/H2O2/BSA, consistent with different modes of radical transfer. More extensive digestion of BSA decreased the radical concentration to levels below those detected with native...

  4. Gas-phase ionization/desolvation processes and their effect on protein charge state distribution under matrix-assisted laser desorption/ionization conditions.

    Science.gov (United States)

    Alves, Sandra; Fournier, Françoise; Afonso, Carlo; Wind, Franck; Tabet, Jean-Claude

    2006-01-01

    The charge state distribution of proteins was studied as a function of experimental conditions, to improve the understanding of the matrix-assisted laser desorption/ionization (MALDI) mechanisms. The relative abundances of the multiply-charged ions appear to be a function of the matrix chosen, the laser fluence and the matrix-to-analyte molar ratio. A correlation is found between the matrix proton affinity and the yield of singly- versus multiply-charged ions. These results are in good agreement with a model in which gas-phase intracluster reactions play a significant role in analyte ion formation. A new model for endothermic desolvation processes in ultraviolet/MALDI is presented and discussed. It is based upon the existence of highly-charged precursor clusters and, complementary to the ion survivor model of Karas et al., assumes that two energy-dependent processes exist: (i) a soft desolvation involving consecutive losses of neutral matrix molecules, leading to a multiply-charged analyte and (ii) hard desolvation leading to a low charge state analyte, by consecutive losses of charged matrix molecules. These desolvations pathways are discussed in terms of kinetically limited processes. The efficiency of the two competitive desolvation processes seems related to the internal energy carried away by clusters during ablation.

  5. Total internal reflection fluorescence anisotropy imaging microscopy: setup, calibration, and data processing for protein polymerization measurements in living cells

    Science.gov (United States)

    Ströhl, Florian; Wong, Hovy H. W.; Holt, Christine E.; Kaminski, Clemens F.

    2018-01-01

    Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring β -actin polymerization in human embryonic kidney cells and in retinal neurons.

  6. Application potential of ATR-FT/IR molecular spectroscopy in animal nutrition: revelation of protein molecular structures of canola meal and presscake, as affected by heat-processing methods, in relationship with their protein digestive behavior and utilization for dairy cattle.

    Science.gov (United States)

    Theodoridou, Katerina; Yu, Peiqiang

    2013-06-12

    Protein quality relies not only on total protein but also on protein inherent structures. The most commonly occurring protein secondary structures (α-helix and β-sheet) may influence protein quality, nutrient utilization, and digestive behavior. The objectives of this study were to reveal the protein molecular structures of canola meal (yellow and brown) and presscake as affected by the heat-processing methods and to investigate the relationship between structure changes and protein rumen degradations kinetics, estimated protein intestinal digestibility, degraded protein balance, and metabolizable protein. Heat-processing conditions resulted in a higher value for α-helix and β-sheet for brown canola presscake compared to brown canola meal. The multivariate molecular spectral analyses (PCA, CLA) showed that there were significant molecular structural differences in the protein amide I and II fingerprint region (ca. 1700-1480 cm(-1)) between the brown canola meal and presscake. The in situ degradation parameters, amide I and II, and α-helix to β-sheet ratio (R_a_β) were positively correlated with the degradable fraction and the degradation rate. Modeling results showed that α-helix was positively correlated with the truly absorbed rumen synthesized microbial protein in the small intestine when using both the Dutch DVE/OEB system and the NRC-2001 model. Concerning the protein profiles, R_a_β was a better predictor for crude protein (79%) and for neutral detergent insoluble crude protein (68%). In conclusion, ATR-FT/IR molecular spectroscopy may be used to rapidly characterize feed structures at the molecular level and also as a potential predictor of feed functionality, digestive behavior, and nutrient utilization of canola feed.

  7. Separation of carbohydrate and protein from wheat for the production of energy and food: conventional and proposed process

    Energy Technology Data Exchange (ETDEWEB)

    Hunwick, R.J.

    1980-09-01

    Historically, wheat has been wet-fractionated to produce starch and gluten, items of value for a broad range of industries as diverse as baking, paper manufacture and sweetener production. In Australia wheat flour has traditionally been the raw material for starch and gluten production with demand for gluten largely dictating starch production. Although this industry is of considerable economic significance in this country, plant throughputs are quite small in a global context. This situation could change dramatically if alcohol derived from wheat were to make a significant contribution to Australia's transport fuel requirements. This paper examines in general terms the impact such a trend could have on starch production in Australia. Traditional flowsheets based upon wheat flour as the raw material are discussed, the most important being the Martin process in which a thick dough is made which is repeatedly washed to liberate starch, bran and solubles as a starch 'milk' from the gluten mass. The starch milk is refined to fractionate its components into relatively pure materials. Recent efforts to improve this technology have been directed towards lowering water consumption mainly to simplify effluent disposal. These have led to the various batter processes which are briefly described. When the object is to produce large quantities of alcohol it is questioned whether it is justified to commence with flour. Whole wheat may be a better feedstock whence wheat could be wet-milled in a manner similar to that employed on a massive scale in North America, in particular for corn (maize). Current corn wet-milling practice is mentioned as an introduction to a summary of novel wet wheat milling flowsheets. Equipment generally used in these flowsheets is described.

  8. Development of C-reactive protein certified reference material NMIJ CRM 6201-b: optimization of a hydrolysis process to improve the accuracy of amino acid analysis.

    Science.gov (United States)

    Kato, Megumi; Kinumi, Tomoya; Yoshioka, Mariko; Goto, Mari; Fujii, Shin-Ichiro; Takatsu, Akiko

    2015-04-01

    To standardize C-reactive protein (CRP) assays, the National Metrology Institute of Japan (NMIJ) has developed a C-reactive protein solution certified reference material, CRM 6201-b, which is intended for use as a primary reference material to enable the SI-traceable measurement of CRP. This study describes the development process of CRM 6201-b. As a candidate material of the CRM, recombinant human CRP solution was selected because of its higher purity and homogeneity than the purified material from human serum. Gel filtration chromatography was used to examine the homogeneity and stability of the present CRM. The total protein concentration of CRP in the present CRM was determined by amino acid analysis coupled to isotope-dilution mass spectrometry (IDMS-AAA). To improve the accuracy of IDMS-AAA, we optimized the hydrolysis process by examining the effect of parameters such as the volume of protein samples taken for hydrolysis, the procedure of sample preparation prior to the hydrolysis, hydrolysis temperature, and hydrolysis time. Under optimized conditions, we conducted two independent approaches in which the following independent hydrolysis and liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) were combined: one was vapor-phase acid hydrolysis (130 °C, 24 h) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, and the other was microwave-assisted liquid-phase acid hydrolysis (150 °C, 3 h) and pre-column derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The quantitative values of the two different amino acid analyses were in agreement within their uncertainties. The certified value was the weighted mean of the results of the two methods. Uncertainties from the value-assignment method, between-method variance, homogeneity, long-term stability, and short-term stability were taken into account in evaluating the uncertainty for a certified value. The certified value and the

  9. Metabolic fingerprinting of high-fat plasma samples processed by centrifugation- and filtration-based protein precipitation delineates significant differences in metabolite information coverage.