Sample records for autoinducer-2 processing protein

  1. The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

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    Diaz, Z.; Xavier, K; Miller, S


    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 (??)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)

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


    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. Analysis of Autoinducer-2 Quorum Sensing in Yersinia pestis (United States)

    Yu, Jing; Madsen, Melissa L.; Carruthers, Michael D.; Phillips, Gregory J.; Kavanaugh, Jeffrey S.; Boyd, Jeff M.; Horswill, Alexander R.


    The autoinducer-2 (AI-2) quorum-sensing system has been linked to diverse phenotypes and regulatory changes in pathogenic bacteria. In the present study, we performed a molecular and biochemical characterization of the AI-2 system in Yersinia pestis, the causative agent of plague. In strain CO92, the AI-2 signal is produced in a luxS-dependent manner, reaching maximal levels of 2.5 μM in the late logarithmic growth phase, and both wild-type and pigmentation (pgm) mutant strains made equivalent levels of AI-2. Strain CO92 possesses a chromosomal lsr locus encoding factors involved in the binding and import of AI-2, and confirming this assignment, an lsr deletion mutant increased extracellular pools of AI-2. To assess the functional role of AI-2 sensing in Y. pestis, microarray studies were conducted by comparing Δpgm strain R88 to a Δpgm ΔluxS mutant or a quorum-sensing-null Δpgm ΔypeIR ΔyspIR ΔluxS mutant at 37°C. Our data suggest that AI-2 quorum sensing is associated with metabolic activities and oxidative stress genes that may help Y. pestis survive at the host temperature. This was confirmed by observing that the luxS mutant was more sensitive to killing by hydrogen peroxide, suggesting a potential requirement for AI-2 in evasion of oxidative damage. We also show that a large number of membrane protein genes are controlled by LuxS, suggesting a role for quorum sensing in membrane modeling. Altogether, this study provides the first global analysis of AI-2 signaling in Y. pestis and identifies potential roles for the system in controlling genes important to disease. PMID:23959719

  4. Autoinducer-2 increases biofilm formation via an ica- and bhp-dependent manner in Staphylococcus epidermidis RP62A. (United States)

    Xue, Ting; Ni, Jingtian; Shang, Fei; Chen, Xiaolin; Zhang, Ming


    Staphylococcus epidermidis has become the most common cause of nosocomial bacteraemia and the principal organism responsible for indwelling medical device -associated infections. Its pathogenicity is mainly due to its ability to form biofilms on the implanted medical devices. Biofilm formation is a quorum-sensing (QS)-dependent process controlled by autoinducers, which are signalling molecules. Here, we investigated the function of the autoinducer-2 (AI-2) QS system, especially the influence of AI-2 on biofilm formation in S. epidermidis RP62A. Results showed that the addition of AI-2 leads to a significant increase in biofilm formation, in contrast with previous studies which showed that AI-2 limits biofilm formation in Staphylococci. We found that AI-2 increases biofilm formation by enhancing the transcription of the ica operon, which is a known component in the AI-2-regulated biofilm pathway. In addition, we first observed that the transcript level of bhp, which encodes a biofilm-associated protein, was also increased following the addition of AI-2. Furthermore, we found that, among the known biofilm regulator genes (icaR, sigB, rbsU, sarA, sarX, sarZ, clpP, agrA, abfR, arlRS, saeRS), only icaR can be regulated by AI-2, suggesting that AI-2 may regulate biofilm formation by an icaR-dependent mechanism in S. epidermidis RP62A.

  5. 变形链球菌UA159Pfs蛋白原核表达纯化及对自诱导分子2合成的影响%Expression and isolation of Streptococcus mutans UA159 S-adenosylhomocysteine nucleosidase protein and observation of its effect on synthesing autoinducer-2

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    黄英; 肖刚; 李芝香; 赵东方; 郭青玉


    Objective To further study the effect of induction molecule autoinducer-2 (AI-2) on the formation of oral biofilm,intending to synthesis Streptococcus mutuans (Sm) AI-2.Methods The prokaryotic expression vector pET21a (+)-pfs of Sm UA159 was constructed by the recombination technology.Then the recombinant vector was transformed into Escherichia coli B121 (DE3),and the expression of Pfs was optimized.The fusion protein was isolated by Ni-chelating column and identified by Western blotting.Then the protein was renatured by dialysis method.S-ribosylhomocysteine (SAH) was catalyzed by Pfs and S-ribosylhomocysteinase(LuxS) to synthesize AI-2.The reaction products induced the luminescence of Vibrio harveyi(Vh) BB170.When the concentration of Pfs protein decreased gradually,the intensity of luminescence emitted by Vh BB170 was observed.Results The recombinant plasmid was confirmed by restriction endonuclease digestion and sequence analysis,which showed that the inserted segment was identical to pfs gene reported by GenBank without nucleotide mutation.The maximum amount of the product expressed was obtained when the isopropylthio-β-D-galactoside (IPTG) concentration was 1 mmol/L,the optimal induction time was 4 h,and the concentration of Pfs was 0.80 g/L.The result of Western blotting showed that the product expressed was Pfs fusion protein.The recombinant Pfs and LuxS synthesized AI-2 in vitro.Up to 5 h,the luminescence intensity of Vh BB170 induced by AI-2 is the strongest [(815 522 ± 73 912) cd].The relative activity of AI-2 and the Pfs concentration are concentration-dependent (P < 0.05),with the rise of the Pfs protein concentration(0-0.38 g/L) the relative activity of AI-2 (0-29.14 ± 1.35) increased gradually(r =0.819,P =0.000).Conclusions The Pfs fusion protein had the biological function,catalyzed the synthesis of the signaling molecule AI-2.The result of the linear correlation analysis showed that the intensity of luminescence emitted by Vh BB170 induced by

  6. D-Galactose as an autoinducer 2 inhibitor to control the biofilm formation of periodontopathogens. (United States)

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


    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.

  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


    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. Autoinducer 2 Signaling via the Phosphotransferase FruA Drives Galactose Utilization by Streptococcus pneumoniae, Resulting in Hypervirulence (United States)

    McAllister, Lauren J.; Chen, Austen; Wang, Hui; Paton, Adrienne W.; Oggioni, Marco R.; McDevitt, Christopher A.


    ABSTRACT 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. PMID:28119473

  9. Identification of poultry meat-derived fatty acids functioning as quorum sensing signal inhibitors of autoinducer-2 (AI-2) (United States)

    Autoinducer-2 (AI-2) is a compound that plays a key role in bacterial cell-to-cell communication (quorum sensing). Previous research has shown certain food matrices inhibit this signaling compound. Using the reporter strain, Vibrio harveyi BB170, quorum sensing inhibitors contained in poultry meat...

  10. Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth (United States)

    Rickard, A.H.; Palmer, R.J.; Blehert, D.S.; Campagna, S.R.; Semmelhack, M.F.; Egland, P.G.; Bassler, B.L.; Kolenbrander, P.E.


    4,5-dihydroxy-2,3-pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). AI-2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by genetic or chemical (synthetic AI-2 in the form of DPD) complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100-fold lower than the detection limit of the currently accepted AI-2 assay. Thus, AI-2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity. ?? 2006 Blackwell Publishing Ltd.

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

    Mohammadi, Mojtaba; Geider, Klaus


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

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

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    Yu Dan


    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.

  13. Autoinducer-2 analogs and electric fields - an antibiotic-free bacterial biofilm combination treatment. (United States)

    Subramanian, Sowmya; Gerasopoulos, Konstantinos; Guo, Min; Sintim, Herman O; Bentley, William E; Ghodssi, Reza


    Bacterial biofilms are a common cause of chronic medical implant infections. Treatment and eradication of biofilms by conventional antibiotic therapy has major drawbacks including toxicity and side effects associated with high-dosage antibiotics. Additionally, administration of high doses of antibiotics may facilitate the emergence of antibiotic resistant bacteria. Thus, there is an urgent need for the development of treatments that are not based on conventional antibiotic therapies. Presented herein is a novel bacterial biofilm combination treatment independent of traditional antibiotics, by using low electric fields in combination with small molecule inhibitors of bacterial quorum sensing - autoinducer-2 analogs. We investigate the effect of this treatment on mature Escherichia coli biofilms by application of an alternating and offset electric potential in combination with the small molecule inhibitor for 24 h using both macro and micro-scale devices. Crystal violet staining of the macro-scale biofilms shows a 46 % decrease in biomass compared to the untreated control. We demonstrate enhanced treatment efficacy of the combination therapy using a high-throughput polydimethylsiloxane-based microfluidic biofilm analysis platform. This microfluidic flow cell is designed to reduce the growth variance of in vitro biofilms while providing an integrated control, and thus allows for a more reliable comparison and evaluation of new biofilm treatments on a single device. We utilize linear array charge-coupled devices to perform real-time tracking of biomass by monitoring changes in optical density. End-point confocal microscopy measurements of biofilms treated with the autoinducer analog and electric fields in the microfluidic device show a 78 % decrease in average biofilm thickness in comparison to the negative controls and demonstrate good correlation with real-time optical density measurements. Additionally, the combination treatment showed 76 % better treatment

  14. Autoinducer-2 influences interactions amongst pioneer colonizing streptococci in oral biofilms. (United States)

    Cuadra-Saenz, Giancarlo; Rao, Dhana L; Underwood, Adam J; Belapure, Sneha A; Campagna, Shawn R; Sun, Zhichao; Tammariello, Steven; Rickard, Alexander H


    Streptococcus gordonii and Streptococcus oralis are among the first bacterial species to colonize clean tooth surfaces. Both produce autoinducer-2 (AI-2): a family of inter-convertible cell-cell signal molecules synthesized by the LuxS enzyme. The overall aim of this work was to determine whether AI-2 alters interspecies interactions between S. gordonii DL1 and S. oralis 34 within dual-species biofilms in flowing human saliva. Based upon AI-2 bioluminescence assays, S. gordonii DL1 produced more AI-2 activity than S. oralis 34 in batch culture, and both were able to remove AI-2 activity from solution. In single-species, saliva-fed flowcell systems, S. oralis 34 formed scant biofilms that were similar to the luxS mutant. Conversely, S. gordonii DL1 formed confluent biofilms while the luxS mutant formed architecturally distinct biofilms that possessed twofold greater biovolume than the wild-type. Supplementing saliva with 0.1-10 nM chemically synthesized AI-2 (csAI-2) restored the S. gordonii DL1 luxS biofilm phenotype to that which was similar to the wild-type; above or below this concentration range, biofilms were architecturally similar to that formed by the luxS mutant. In dual-species biofilms, S. gordonii DL1 was always more abundant than S. oralis 34. Compared with dual-species, wild-type biofilms, the biovolume occupied by S. oralis 34 was reduced by greater than sevenfold when neither species produced AI-2. The addition of 1 nM csAI-2 to the dual-species luxS-luxS mutant biofilms re-established the biofilm phenotype to resemble that of the wild-type pair. Thus, this work demonstrates that AI-2 can alter the biofilm structure and composition of pioneering oral streptococcal biofilms. This may influence the subsequent succession of other species into oral biofilms and the ecology of dental plaque.

  15. Determination of autoinducer-2 in biological samples by high-performance liquid chromatography with fluorescence detection using pre-column derivatization. (United States)

    Song, Xiang-Ning; Qiu, Hai-Bin; Xiao, Xiang; Cheng, Yuan-Yuan; Li, Wen-Wei; Sheng, Guo-Ping; Li, Xiao-Yan; Yu, Han-Qing


    Autoinducer-2 (AI-2), as a small-molecular-weight organic molecule secreted and perceived by various bacteria, enables intra- and inter-species communications. Quantitative determination of AI-2 is essential for exploring the bacterial AI-2-related physiological and biochemical processes. However, current strategies for sensitive detection of AI-2 require sophisticated instruments and complicated procedures. In this work, on the basis of the derivatization of AI-2 with 2,3-diaminonaphthalene, a simple, sensitive and cost-effective high-performance liquid chromatography with fluorescence detector (HPLC-FLD) method is developed for the quantitative detection of AI-2. Under the optimized conditions, this method had a broad linear range of 10-14,000 ng/ml (R(2)=0.9999), and a low detection limit of 1.0 ng/ml. Furthermore, the effectiveness of this approach was further validated through measuring the AI-2 concentrations in the cell-free culture supernatants of both Escherichia coli and Vibrio harveyi.

  16. New Insights into Autoinducer-2 Signaling as a Virulence Regulator in a Mouse Model of Pneumonic Plague (United States)

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


    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

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

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    Steven E A Christiaen

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

  18. Autoinducer-2-like activity on vegetable produce and its potential involvement in bacterial biofilm formation on tomatoes. (United States)

    Lu, Lingeng; Hume, Michael E; Pillai, Suresh D


    Quorum sensing employing autoinducer molecules is a strategy used by bacterial populations to coordinately modulate their response to environmental stresses and host defense mechanisms. The objectives of this study were to determine the levels of autoinducer-2 (AI-2)-like activity on selected vegetable produce and determine whether AI-2-like molecules can promote E. coli O157:H7 biofilm formation on tomatoes. Twelve different fruit and vegetable produce samples were screened for AI-2-like activity using autoinducer sensing V. harveyi biosensor strains. All samples except strawberries showed AI-2 activity albeit at varying levels, with eggplant having the highest levels. Tomatoes, when stored at 4 degrees C for 9 days, showed increasing levels of heterotrophic bacterial populations as compared to AI-2-like activity levels, which fluctuated. Rinses from Roma tomato surfaces that were stored at refrigeration temperature for up to 9 days caused a significant increase (1.8-3.6-fold as compared to the negative controls) in biofilm formation by luxS mutant (non AI-2 producing) generic E. coli and E. coli O157:H7 strains using a micro-titer plate-based biofilm assay. These results suggest that AI-2-like activity, which is present on the surfaces of tomatoes, has the potential to enhance the production of bacterial biofilms.

  19. Production of autoinducer-2 by aerobic endospore-forming bacteria isolated from the West African fermented foods. (United States)

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


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

  20. Identification of poultry meat-derived fatty acids functioning as quorum sensing signal inhibitors to autoinducer-2 (AI-2). (United States)

    Widmer, K W; Soni, K A; Hume, M E; Beier, R C; Jesudhasan, P; Pillai, S D


    Autoinducer-2 (AI-2) is a compound that plays a key role in bacterial cell-to-cell communication (quorum sensing). Previous research has shown certain food matrices inhibit this signaling compound. Using the reporter strain, Vibrio harveyi BB170, quorum-sensing inhibitors contained in poultry meat wash (PMW) samples were characterized by molecular weight and hydrophobic properties using liquid chromatography systems. Most fractions that demonstrated AI-2 inhibition were 13.7 kDa or less, and had hydrophobic properties. Hexane was used to extract inhibitory compounds from a PMW preparation and the extract was further separated by gas chromatography (GC). Several fatty acids were identified and quantified. Linoleic acid, oleic acid, palmitic acid, and stearic acid were each tested for inhibition at 0.1, 1, and 10 mM concentrations. All samples expressed AI-2 inhibition (ranging from approximately 25% to 99%). Fatty acids, combined in concentrations equivalent to those determined by GC analysis, expressed inhibition at 59.5%, but higher combined concentrations (10- and 100-fold) had inhibition at 84.4% and 69.5%, respectively. The combined fatty acids (100-fold) did not demonstrate a substantial decrease in colony plate counts, despite presenting high AI-2 inhibition. These fatty acids, through modulating quorum sensing by inhibition, may offer a unique means to control foodborne pathogens and reduce microbial spoilage.

  1. A Host-Produced Autoinducer-2 Mimic Activates Bacterial Quorum Sensing. (United States)

    Ismail, Anisa S; Valastyan, Julie S; Bassler, Bonnie L


    Host-microbial symbioses are vital to health; nonetheless, little is known about the role crosskingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote interspecies bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering crosskingdom signaling and host-bacterial symbioses.

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

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


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

  3. Process for protein PEGylation. (United States)

    Pfister, David; Morbidelli, Massimo


    PEGylation is a versatile drug delivery technique that presents a particularly wide range of conjugation chemistry and polymer structure. The conjugated protein can be tuned to specifically meet the needs of the desired application. In the area of drug delivery this typically means to increase the persistency in the human body without affecting the activity profile of the original protein. On the other hand, because of the high costs associated with the production of therapeutic proteins, subsequent operations imposed by PEGylation must be optimized to minimize the costs inherent to the additional steps. The closest attention has to be given to the PEGylation reaction engineering and to the subsequent purification processes. This review article focuses on these two aspects and critically reviews the current state of the art with a clear focus on the development of industrial scale processes which can meet the market requirements in terms of quality and costs. The possibility of using continuous processes, with integration between the reaction and the separation steps is also illustrated.

  4. Bifidobacterium spp. influences the production of autoinducer-2 and biofilm formation by Escherichia coli O157:H7. (United States)

    Kim, Younghoon; Lee, Jae Won; Kang, Seo-Gu; Oh, Sejong; Griffiths, Mansel W


    The effect of Bifidobacterium spp. on the production of quorum-sensing (QS) signals and biofilm formation by enterohemorrhagic Escherichia coli (EHEC) O157:H7 was investigated. In an AI-2 bioassay, cell extracts of Bifidobacterium longum ATCC 15707 resulted in a 98-fold reduction in AI-2 activity in EHEC O157:H7 as well as in the Vibrio harveyi reporter strain, even though they did not inhibit the growth of EHEC O157:H7. In addition, they resulted in a 36% reduction in biofilm formation by the organism. Consistently, the virulence of EHEC O157:H7 was significantly attenuated by the presence of cell extracts of B. longum ATCC 15707 in the Caenorhabditis elegans nematode in vivo model. By a proteome analysis using two dimensional electrophoresis (2-DE), we determined that seven proteins including formation of iron-sulfur protein (NifU), thiol:disulfide interchange protein (DsbA), and flagellar P-ring protein (FlgI) were differentially regulated in the EHEC O157:H7 when supplemented with cell extracts of B. longum ATCC 15707. Taken together, these findings propose a novel function of a dairy adjunct in repressing the virulence of EHEC O157:H7.

  5. Production of autoinducer 2 in Salmonella enterica serovar Thompson contributes to its fitness in chickens but not on cilantro leaf surfaces. (United States)

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


    Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size of the S. enterica serovar Thompson parental strain was significantly higher than that of its LuxS(-) mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS(-) strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of S. enterica serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS(-) mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS(-) mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to S. enterica in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of S. enterica in these environments.

  6. Quorum-sensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells. (United States)

    Vidal, Jorge E; Howery, Kristen E; Ludewick, Herbert P; Nava, Porfirio; Klugman, Keith P


    Streptococcus pneumoniae forms organized biofilms in the human upper respiratory tract that may play an essential role in both persistence and acute respiratory infection. However, the production and regulation of biofilms on human cells is not yet fully understood. In this work, we developed a bioreactor with living cultures of human respiratory epithelial cells (HREC) and a continuous flow of nutrients, mimicking the microenvironment of the human respiratory epithelium, to study the production and regulation of S. pneumoniae biofilms (SPB). SPB were also produced under static conditions on immobilized HREC. Our experiments demonstrated that the biomass of SPB increased significantly when grown on HREC compared to the amount on abiotic surfaces. Additionally, pneumococcal strains produced more early biofilms on lung cells than on pharyngeal cells. Utilizing the bioreactor or immobilized human cells, the production of early SPB was found to be regulated by two quorum-sensing systems, Com and LuxS/AI-2, since a mutation in either comC or luxS rendered the pneumococcus unable to produce early biofilms on HREC. Interestingly, while LuxS/autoinducer 2 (AI-2) regulated biofilms on both HREC and abiotic surfaces, Com control was specific for those structures produced on HREC. The biofilm phenotypes of strain D39-derivative ΔcomC and ΔluxS QS mutants were reversed by genetic complementation. Of note, SPB formed on immobilized HREC and incubated under static conditions were completely lysed 24 h postinoculation. Biofilm lysis was also regulated by the Com and LuxS/AI-2 quorum-sensing systems.

  7. In Helicobacter pylori auto-inducer-2, but not LuxS/MccAB catalysed reverse transsulphuration, regulates motility through modulation of flagellar gene transcription

    Directory of Open Access Journals (Sweden)

    Doherty Neil


    Full Text Available Abstract Background LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2; hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB, synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only. Results We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the ΔluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2. In contrast, exogenously added cysteine could not restore motility to the ΔluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the ΔluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the ΔluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the ΔluxSHp mutant restored flagellar gene transcription, and the number and length of flagella. Conclusions Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2.

  8. Process for Encapsulating Protein Crystals (United States)

    Morrison, Dennis R.; Mosier, Benjamin


    A process for growing protein crystals encapsulated within membranes has been invented. This process begins with the encapsulation of a nearly saturated aqueous protein solution inside semipermeable membranes to form microcapsules. The encapsulation is effected by use of special formulations of a dissolved protein and a surfactant in an aqueous first liquid phase, which is placed into contact with a second, immiscible liquid phase that contains one or more polymers that are insoluble in the first phase. The second phase becomes formed into the semipermeable membranes that surround microglobules of the first phase, thereby forming the microcapsules. Once formed, the microcapsules are then dehydrated osmotically by exposure to a concentrated salt or polymer solution. The dehydration forms supersaturated solutions inside the microcapsules, thereby enabling nucleation and growth of protein crystals inside the microcapsules. By suitable formulation of the polymer or salt solution and of other physical and chemical parameters, one can control the rate of transport of water out of the microcapsules through the membranes and thereby create physicochemical conditions that favor the growth, within each microcapsule, of one or a few large crystals suitable for analysis by x-ray diffraction. The membrane polymer can be formulated to consist of low-molecular-weight molecules that do not interfere with the x-ray diffraction analysis of the encapsulated crystals. During dehydration, an electrostatic field can be applied to exert additional control over the rate of dehydration. This protein-crystal-encapsulation process is expected to constitute the basis of protein-growth experiments to be performed on the space shuttle and the International Space Station. As envisioned, the experiments would involve the exposure of immiscible liquids to each other in sequences of steps under microgravitational conditions. The experiments are expected to contribute to knowledge of the precise

  9. 变形链球菌 UA159合成自诱导分子2及其影响因素的实验研究%In vitro biosynthesis of autoinducer 2 of Streptococcus mutans UA1 59 and observation of the influence factors

    Institute of Scientific and Technical Information of China (English)

    李芝香; 肖刚; 黄英; 赵东方; 刘飞; 刘阳; 郭青玉


    目的:原核表达纯化变形链球菌(S.mutans)UA159 LuxS 蛋白,体外合成有活性的自诱导分子2(AI-2)并观察其合成的影响因素。方法:用基因重组的方法构建表达载体 pET21 a(+)-luxS,在大肠杆菌(E.coli)BL21(DE3)中诱导表达 S-核糖基高半胱氨酸酶(LuxS)融合蛋白,镍柱亲和层析纯化、蛋白质印迹法分析鉴定 LuxS 蛋白,透析复性。纯化的 LuxS 蛋白和 S-腺苷高半胱氨酸核苷酶(Pfs)蛋白催化 S-腺苷 L 高半胱氨酸[SAH]合成 AI-2,哈氏弧菌 BB170检测 AI-2活性,并观察 LuxS 蛋白浓度、pH、氟化钠对 AI-2合成的影响。结果:与对照组相比,随着 LuxS 蛋白浓度的升高,体外合成的 AI-2活性增大(P <0.001);当 pH 介于6~10之间时,AI-2活性最强,超出此 pH 范围,AI-2活性明显降低(P <0.001);当氟化钠浓度≥0.3%时,AI-2活性显著降低(P <0.05)。结论:利用基因工程技术可以合成具有生物活性的 S.mutans UA159 AI-2;AI-2合成的最适 pH 在6~10之间;氟化钠浓度≥0.3%对 AI-2的合成有抑制作用。%Objective:To synthesize autoinducer-2 by the clone and prokaryotic expression of Streptococcus mutans(S.mutans)UAl59 luxS gene and to observe the influence factors.Methods:The expression vector pET21 a(+)-luxS of S.mutans UAl59 was transformed into Escheriehia coli BL2l(DE3).The S-ribosylhomocysteinase(Luxs)expression was induced by IPTG.The His tag fusion protein was isolated by Ni-chelating column and identified by Western blotting.Finally the protein was renatured by dialysis method.S-ribosylhomo-cysteine (SAH)was catalyzed by s-adenosylhomocysteine nucleosidas (Pfs)and LuxS,and then AI-2 was syntheszed.The AI-2 activi-ty was examined by luminescence of Vibrio harveyi BB1 70 when the concentration of LuxS protein or pH(4 -1 2)or the concentration of sodium fluoride was changed in reaction mixes of AI-2

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

    NARCIS (Netherlands)

    Salazar Villanea, Sergio


    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 am

  11. A Signal Processing Method to Explore Similarity in Protein Flexibility

    Directory of Open Access Journals (Sweden)

    Simina Vasilache


    Full Text Available Understanding mechanisms of protein flexibility is of great importance to structural biology. The ability to detect similarities between proteins and their patterns is vital in discovering new information about unknown protein functions. A Distance Constraint Model (DCM provides a means to generate a variety of flexibility measures based on a given protein structure. Although information about mechanical properties of flexibility is critical for understanding protein function for a given protein, the question of whether certain characteristics are shared across homologous proteins is difficult to assess. For a proper assessment, a quantified measure of similarity is necessary. This paper begins to explore image processing techniques to quantify similarities in signals and images that characterize protein flexibility. The dataset considered here consists of three different families of proteins, with three proteins in each family. The similarities and differences found within flexibility measures across homologous proteins do not align with sequence-based evolutionary methods.

  12. Retinoblastoma protein: a central processing unit. (United States)

    Poznic, M


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

  13. Retinoblastoma protein: a central processing unit

    Indian Academy of Sciences (India)

    M Poznic


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

  14. Protein import into plant mitochondria: signals, machinery, processing, and regulation. (United States)

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


    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.

  15. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M


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

  16. Improved corn protein (zein) extrusion processing (United States)

    Melt processing using a single and twin screw extruder has been carried out on zein where extrusion temperatures were varied between 100ºC and 300ºC. In addition, melt reprocessing (up to seven times) of zein was undertaken using a single screw extruder. Differential scanning calorimetry (DSC) and t...

  17. [New procedure for protein spinning: the hydrodynamic process]. (United States)

    Castaigne, F; Liber, E; Carbillet, L; Boulet, M; Riel, R R


    In this text, we describe a new protein spinning process called hydrodynamic process. Parameters which are related to production of fibers and which can influence diameter and texture are explained extensively. In this process, a spinning dope is extruded through a spinnerette in a moving coagulation bath in which the fibers are coagulated, stretched and carried.

  18. Integrated process for high conversion and high yield protein PEGylation. (United States)

    Pfister, David; Morbidelli, Massimo


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

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

    NARCIS (Netherlands)

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


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

  20. Thermoplastic processing of proteins for film formation--a review. (United States)

    Hernandez-Izquierdo, V M; Krochta, J M


    Increasing interest in high-quality food products with increased shelf life and reduced environmental impact has encouraged the study and development of edible and/or biodegradable polymer films and coatings. Edible films provide the opportunity to effectively control mass transfer among different components in a food or between the food and its surrounding environment. The diversity of proteins that results from an almost limitless number of side-chain amino-acid sequential arrangements allows for a wide range of interactions and chemical reactions to take place as proteins denature and cross-link during heat processing. Proteins such as wheat gluten, corn zein, soy protein, myofibrillar proteins, and whey proteins have been successfully formed into films using thermoplastic processes such as compression molding and extrusion. Thermoplastic processing can result in a highly efficient manufacturing method with commercial potential for large-scale production of edible films due to the low moisture levels, high temperatures, and short times used. Addition of water, glycerol, sorbitol, sucrose, and other plasticizers allows the proteins to undergo the glass transition and facilitates deformation and processability without thermal degradation. Target film variables, important in predicting biopackage performance under various conditions, include mechanical, thermal, barrier, and microstructural properties. Comparisons of film properties should be made with care since results depend on parameters such as film-forming materials, film formulation, fabrication method, operating conditions, testing equipment, and testing conditions. Film applications include their use as wraps, pouches, bags, casings, and sachets to protect foods, reduce waste, and improve package recyclability.

  1. Inhalable dust and protein exposure in soybean processing plants. (United States)

    Spies, Adri; Rees, David; Fourie, Anna M; Wilson, Kerry S; Harris-Roberts, Joanne; Robinson, Edward


    Little is known about inhalable dust concentrations in soybean processing plants in southern Africa. This project measured inhalable dust in soybean plants in the region and correlated dust measurements with total protein and soy trypsin inhibitor. Sixty-four personal inhalable dust measurements were taken in three processing plants. Levels of total protein and soy trypsin inhibitor were determined in only two of the three plants. Correlations between inhalable dust, total protein and trypsin inhibitor were determined for 44 of 64 samples. In plants' production areas, inhalable dust levels were 0.24-35.02 mg/m3 (median 2.58 mg/m3). Total protein and soy trypsin inhibitor levels were 29.41-448.82 microg/m3 (median 90.09 microg/m3) and 0.05-2.58 microg/m3 (median 0.07 microg/m3), respectively. No statistically significant correlations between presence of inhalable dust and soy trypsin inhibitor were found. Total protein and soy trypsin inhibitor were better correlated. This study indicates that total protein might be a good proxy for soybean specific protein concentrations in soybean processing plants.

  2. RNA-processing protein TDP-43 regulates FOXO-dependent protein quality control in stress response.

    Directory of Open Access Journals (Sweden)

    Tao Zhang


    Full Text Available Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43 is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.

  3. [Processing and Modification of Recombinant Spider Silk Proteins]. (United States)

    Liu, Bin; Wang, Tao; Liu, Xiaobing; Luo, Yongen


    Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein.

  4. Intrinsic and extrinsic negative regulators of nuclear protein transport processes


    Sekimoto, Toshihiro; Yoneda, Yoshihiro


    The nuclear–cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclea...

  5. Predicting Protein Subcellular Location Using Digital Signal Processing

    Institute of Scientific and Technical Information of China (English)

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


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

  6. Predicting protein subcellular location using digital signal processing. (United States)

    Pan, Yu-Xi; Li, Da-Wei; Duan, Yun; Zhang, Zhi-Zhou; Xu, Ming-Qing; Feng, Guo-Yin; He, Lin


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

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

    Institute of Scientific and Technical Information of China (English)


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

  8. Piezo proteins: regulators of mechanosensation and other cellular processes. (United States)

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G


    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature.

  9. Downstream processing of biopharmaceutical proteins produced in plants (United States)

    Buyel, Johannes Felix; Fischer, Rainer


    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. PMID:24637706

  10. Processing of heme and heme-containing proteins by bacteria. (United States)

    Stojiljkovic, Igor; Perkins-Balding, Donna


    An extensive amount of new knowledge on bacterial systems involved in heme processing has been accumulated in the last 10 years. We discuss common themes in heme transport across bacterial outer and inner membranes, emphasizing proteins and mechanisms involved. The processing of heme in the bacterial cytoplasm is extensively covered, and a new hypothesis about the fate of heme in the bacterial cell is presented. Auxiliary genes involved in heme utilization, i.e., TonB, proteases, proteins involved in heme storage and pigmentation, as well as genes involved in regulation of heme assimilation are reviewed.

  11. 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)


    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.

  12. Plasma proteins as biomarkers of the aging process. (United States)

    Vranckx, R; Savu, L; Lambert, N; de Conchard, G V; Grosse, R; Mourey, M S; Corman, B


    This study was designed to characterize the rat serum proteins as biomarkers of the normal aging process. Crossed immunoelectrophoresis or electroimmunodiffusion quantitation of proteins was performed in rats aged 6, 12, 24, and 30 mo. Selection of healthy animals was based on confrontation of crossed immunoelectrophoresis patterns with those of experimentally inflamed young adults and with individual anatomopathological data. Convergence of inflammatory patterns and severe histological lesions was the exclusion criterion. Senescence-induced decrease was demonstrated for eight proteins [negative senescence reactants (SRs-)] and increase for six proteins [positive SRs (SRs+)]. Most SRs belonged to the class of proteins responsive to acute inflammation [acute phase reactants (APRs)]. One SR+, the thyroxine-binding globulin, a high-affinity thyroid hormone binder, emerged as a particularly reliable senescence biomarker, showing the highest aging-related variation (8-fold increase from 6 to 30 mo) and not belonging to the APR class. Chronic treatment with perindopril, an angiotensin I-converting enzyme inhibitor used in heart and renal disease therapy, significantly enhanced thyroxine-binding capacity, possibly by preventing age-related alterations of serum lipids. Serum protein patterns prove valuable both as indexes for selecting aging animals free from superimposed pathologies and as parameters of senescence-induced changes in protein biosynthesis.

  13. Extraction and downstream processing of plant-derived recombinant proteins. (United States)

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


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

  14. High pressure-low temperature processing of food proteins. (United States)

    Dumay, Eliane; Picart, Laetitia; Regnault, Stéphanie; Thiebaud, Maryse


    High pressure-low temperature (HP-LT) processing is of interest in the food field in view of: (i) obtaining a "cold" pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP-LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP-LT have been investigated on protein models such as (i) beta-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP-LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of approximately 2% beta-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles.

  15. Process considerations for protein engineering of ω-Transaminase

    DEFF Research Database (Denmark)

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

    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...

  16. Influence of processing on functionality of milk and dairy proteins. (United States)

    Augustin, Mary Ann; Udabage, Punsandani


    The inherent physical functionality of dairy ingredients makes them useful in a range of food applications. These functionalities include their solubility, water binding, viscosity, gelation, heat stability, renneting, foaming, and emulsifying properties. The suitability of dairy ingredients for an application can be further tailored by altering the structure of the proteins using appropriate processes. The processes discussed include physical modification (heat treatment, acidification, addition of mineral slats, homogenization, and shear), enzymatic modification (renneting, hydrolysis, and transglutamination), and chemical modification (use of chemical agents and the Maillard reaction). Emerging food processes (high pressure and ultrasound) are also discussed. The challenges for using dairy ingredients for the delivery of nutrients and bioactive components, while maintaining physical functionality, are also highlighted. There is a need for continued research into the fundamental aspects of milk proteins and their responses to various stresses for further differentiation of milk products and for the delivery of ingredients with consistent quality for target applications.

  17. Role of preterminal protein processing in adenovirus replication. (United States)

    Webster, A; Leith, I R; Nicholson, J; Hounsell, J; Hay, R T


    Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.

  18. Optimization of protein electroextraction from microalgae by a flow process. (United States)

    Coustets, Mathilde; Joubert-Durigneux, Vanessa; Hérault, Josiane; Schoefs, Benoît; Blanckaert, Vincent; Garnier, Jean-Pierre; Teissié, Justin


    Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size.

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

    Directory of Open Access Journals (Sweden)

    Nuring Wulansari


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

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


    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.

  1. Amyloid Precursor Protein Processing in Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Adwait BHADBHADE


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

  2. [Proteins of human milk involved in immunological processes]. (United States)

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


    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  3. Proteins of human milk involved in immunological processes 

    Directory of Open Access Journals (Sweden)

    Jolanta Lis


    Full Text Available Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  4. The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb. (United States)

    Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye


    The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.

  5. Exploring novel food proteins and processing technologies : a case study on quinoa protein and high pressure –high temperature processing

    NARCIS (Netherlands)

    Avila Ruiz, Geraldine


    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, th

  6. Increasing importance of protein flexibility in designing biocatalytic processes

    Directory of Open Access Journals (Sweden)

    Joyeeta Mukherjee


    Full Text Available Enzymes require some flexibility for catalysis. Biotechnologists prefer stable enzymes but often this stabilization comes at the cost of reduced efficiency. Enzymes from thermophiles have low flexibility but poor catalytic rates. Enzymes from psychrophiles are less stable but show good catalytic rates at low temperature. In organic solvents enzymes perform poorly as the prior drying makes the enzyme molecules very rigid. Adding water or increasing reaction temperature improves flexibility and catalytic rates. In case of hydrolases, flexibility and enantioselectivity have interdependence. Understanding the complex role of protein flexibility in biocatalysis can help in designing biotechnological processes.

  7. The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis. (United States)

    Minamino, Tohru; Morimoto, Yusuke V; Kinoshita, Miki; Aldridge, Phillip D; Namba, Keiichi


    For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI(6)FliJ complex is structurally similar to the α(3)β(3)γ complex of F(O)F(1)-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI(6)FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly.

  8. Betaine suppressed Aβ generation by altering amyloid precursor protein processing. (United States)

    Liu, Xiu-Ping; Qian, Xiang; Xie, Yue; Qi, Yan; Peng, Min-Feng; Zhan, Bi-Cui; Lou, Zheng-Qing


    Betaine was an endogenous catabolite of choline, which could be isolated from vegetables and marine products. Betaine could promote the metabolism of homocysteine in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver in clinic. Recent findings shown that Betaine rescued neuronal damage due to homocysteine induced Alzheimer's disease (AD) like pathological cascade, including tau hyperphosphorylation and amyloid-β (Aβ) deposition. Aβ was derived from amyloid precursor protein (APP) processing, and was a triggering factor for AD pathological onset. Here, we demonstrated that Betaine reduced Aβ levels by altering APP processing in N2a cells stably expressing Swedish mutant of APP. Betaine increased α-secretase activity, but decreased β-secretase activity. Our data indicate that Betaine might play a protective role in Aβ production.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  10. Persistence of pathogenic prion protein during simulated wastewater treatment processes (United States)

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


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

  11. Helicobacter pylori protein oxidation influences the colonization process. (United States)

    Godlewska, Renata; Dzwonek, Artur; Mikuła, Michał; Ostrowski, Jerzy; Pawłowski, Marcin; Bujnicki, Janusz M; Jagusztyn-Krynicka, Elzbieta K


    Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of membrane and exported proteins. Here we examined the role of DsbI protein in Helicobacter pylori pathogenesis and demonstrated that a dsbI mutant impaired in disulfide bond formation revealed a greatly reduced ability to colonize mice gastric mucosa.

  12. Pea proteins for piglets: effects on digestive processes.

    NARCIS (Netherlands)

    le Guen, M.P.


    White-flowered pea ( Pisum sativum ) contains 20 to 25% crude protein (Nx6.25). The pea proteins consist in globulins (60%) and albumins (25%). Because the pea albumin proteins have biological activities due to their metabolic roles in the seed, some of them are called antinutritional factors (ANFs)

  13. Identification of Giardia lamblia DHHC proteins and the role of protein S-palmitoylation in the encystation process. (United States)

    Merino, María C; Zamponi, Nahuel; Vranych, Cecilia V; Touz, María C; Rópolo, Andrea S


    Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

  14. Soybean bio-refinery platform: enzymatic process for production of soy protein concentrate, soy protein isolate and fermentable sugar syrup. (United States)

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


    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.

  15. Zein synthesis and processing on zein protein body membranes. [Maize proteins

    Energy Technology Data Exchange (ETDEWEB)

    Burr, F A


    The storage protein of maize, zein, is translated from messenger RNA on ribosomes bound to the outer membrane of the zein protein bodies. No other proteins appear to be made on this membrane. Before zein is transported through the protein body membrane it undergoes at least two post-translational modifications, which are discussed.

  16. Production of personalized protein microarrays : optimized production of protein microarrays and the establishment of processes for the representation of protein conformations that occur in individual patients



    Despite remarkable progress in understanding biology and disease at the level of nucleic acids, insights into the relevant biochemical processes frequently remain preliminary, since much regulation and activity occurs at the protein level through control of gene expression and variations of protein conformation. In particular, the effect of such variations on protein interactions is critical for a better description of biology and disease. Protein microarray technology provides a means to suc...

  17. Understanding leaf membrane protein extraction to develop a food-grade process. (United States)

    Tamayo Tenorio, Angelica; Boom, Remko M; van der Goot, Atze Jan


    Leaf membrane proteins are an underutilised protein fraction for food applications. Proteins from leaves can contribute to a more complete use of resources and help to meet the increasing protein demand. Leaf protein extraction and purification is applied by other disciplines, such as proteomics. Therefore, this study analysed proteomic extraction methods for membrane proteins as an inspiration for a food-grade alternative process. Sugar beet leaves were extracted with two proteomic protocols: solvent extraction and Triton X-114 phase partitioning method. Extraction steps contributed to protein purity and/or to selective fractionation, enabling the purification of specific proteins. It was observed that membrane proteins distributed among different solvents, buffers and solutions used due to their physicochemical heterogeneity. This heterogeneity does not allow a total membrane protein extraction by a unique method or even combinations of processing steps, but it enables the creation of different fractions with different physicochemical properties useful for food applications.

  18. Multimodal chromatography: an efficient tool in downstream processing of proteins. (United States)

    Kallberg, Kristian; Johansson, Hans-Olof; Bulow, Leif


    Chromatography has become an indispensable tool for the purification of proteins. Since the regulatory demands on protein purity are expected to become stricter, the need for generating improved resins for chromatographic separations has increased. More advanced scientific investigations of protein structure/function relationships, in particular, have also been a driving force for generating more sophisticated chromatographic materials for protein separations. As a consequence, the development of alternative chromatographic strategies has been very rapid during the past decade and several new ligands have been designed and explored both in the laboratory and in large-scale industrial settings. This review describes some of these efforts using multimodal chromatography, where two or more physicochemical properties are used to enhance the specificity of the interactions between the protein and the ligand on the chromatographic matrix. In addition to experimental studies, computer modeling of ligand-protein binding has improved the design of ligands for protein recognition. The use of descriptors as well as in silico docking methods have been implemented to design multimodal resins in several instances.

  19. Virus host protein interaction network analysis reveals that the HEV ORF3 protein may interrupt the blood coagulation process.

    Directory of Open Access Journals (Sweden)

    Yansheng Geng

    Full Text Available Hepatitis E virus (HEV is endemic worldwide and a major cause of acute liver disease in developing countries. However, the molecular mechanisms of liver pathology and clinical disease are not well understood for HEV infection. Open reading frame 3 (ORF3 of HEV encodes a small phosphoprotein, which is assumed to be involved in liver pathology and clinical disease. In this study, the interactions between the HEV ORF3 protein and human proteins were investigated using a stringent, high-throughput yeast two-hybrid (Y2H analysis. Thirty two proteins were shown to interact with genotype 1 ORF3, 28 of which have not been reported previously. These novel interactions were evaluated by coimmunoprecipitation of protein complexes from transfected cells. We found also that the ORF3 proteins of genotype 4 and rabbit HEV interacted with all of the human proteins identified by the genotype 1 ORF3 protein. However, the putative ORF3 protein derived from avian HEV did not interact with the majority of these human proteins. The identified proteins were used to infer an overall interaction map linking the ORF3 protein with components of the host cellular networks. Analysis of this interaction map, based on functional annotation with the Gene Ontology features and KEGG pathways, revealed an enrichment of host proteins involved in complement coagulation, cellular iron ion homeostasis and oxidative stress. Additional canonical pathway analysis highlighted the enriched biological pathways relevant to blood coagulation and hemostasis. Consideration of the clinical manifestations of hepatitis E reported previously and the results of biological analysis from this study suggests that the ORF3 protein is likely to lead to an imbalance of coagulation and fibrinolysis by interacting with host proteins and triggering the corresponding pathological processes. These results suggest critical approaches to further study of the pathogenesis of the HEV ORF3 protein.

  20. Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization. (United States)

    Heiss, Silvia; Puxbaum, Verena; Gruber, Clemens; Altmann, Friedrich; Mattanovich, Diethard; Gasser, Brigitte


    Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

  1. Dynamical analysis of yeast protein interaction network during the sake brewing process. (United States)

    Mirzarezaee, Mitra; Sadeghi, Mehdi; Araabi, Babak N


    Proteins interact with each other for performing essential functions of an organism. They change partners to get involved in various processes at different times or locations. Studying variations of protein interactions within a specific process would help better understand the dynamic features of the protein interactions and their functions. We studied the protein interaction network of Saccharomyces cerevisiae (yeast) during the brewing of Japanese sake. In this process, yeast cells are exposed to several stresses. Analysis of protein interaction networks of yeast during this process helps to understand how protein interactions of yeast change during the sake brewing process. We used gene expression profiles of yeast cells for this purpose. Results of our experiments revealed some characteristics and behaviors of yeast hubs and non-hubs and their dynamical changes during the brewing process. We found that just a small portion of the proteins (12.8 to 21.6%) is responsible for the functional changes of the proteins in the sake brewing process. The changes in the number of edges and hubs of the yeast protein interaction networks increase in the first stages of the process and it then decreases at the final stages.

  2. DSCG binding protein and process for preparing same

    Energy Technology Data Exchange (ETDEWEB)

    Pecht, I.; Mazurek, N.


    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. 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.


    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)

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

    Institute of Scientific and Technical Information of China (English)


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

  5. Non-conventional approaches to food processing in CELSS. I - Algal proteins: Characterization and process optimization (United States)

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


    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.

  6. Non-conventional approaches to food processing in CELSS, 1. Algal proteins: Characterization and process optimization (United States)

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


    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.

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

    NARCIS (Netherlands)

    Poel, van der A.F.B.


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

  8. Characteristics of extraction and functionality of protein from tomato pomace produced with different industrial processing methods (United States)

    The seeds from tomato pomace, a by-product of tomato processing, contains valuable but underutilized protein with unique functional properties. The objectives of this research were to study the impact of industrial hot and cold break tomato processing on protein extraction from defatted tomato seeds...

  9. Real-time processing of interferograms for monitoring protein crystal growth on the Space Station (United States)

    Choudry, A.; Dupuis, N.


    The possibility of using microscopic interferometric techniques to monitor the growth of protein crystals on the Space Station is studied. Digital image processing techniques are used to develop a system for the real-time analysis of microscopic interferograms of nucleation sites during protein crystal growth. Features of the optical setup and the image processing system are discussed and experimental results are presented.

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


    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.

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

    CERN Document Server

    Bier, M


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

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

    Institute of Scientific and Technical Information of China (English)


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

  13. N-terminal Protein Processing: A Comparative Proteogenomic Analysis*


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


    N-terminal methionine excision (NME) and N-terminal acetylation (NTA) are two of the most common protein post-translational modifications. NME is a universally conserved activity and a highly specific mechanism across all life forms. NTA is very common in eukaryotes but occurs rarely in prokaryotes. By analyzing data sets from yeast, mammals and bacteria (including 112 million spectra from 57 bacterial species), the largest comparative proteogenomics study to date, it is shown that previous a...

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


    Poel, van der, A.F.B.


    In animal production, feeding has an important impact on productivity and health of animals and feed composition is known to influence protein and energy metabolism directly. For monogastric animals complete diets are manufactured in which feed ingredients are used to supply the energy yielding and other nutrients. The common bean ( Phaseolus vulgaris L.) is such an ingredient.In common beans ( Phaseolus vulgaris L.) the supply of nutrients is often lower than is expected from its chemical an...

  15. Ribosome recycling: An essential process of protein synthesis. (United States)

    Kiel, Michael C; Kaji, Hideko; Kaji, Akira


    A preponderance of textbooks outlines cellular protein synthesis (translation) in three basic steps: initiation, elongation, and termination. However, researchers in the field of translation accept that a vital fourth step exists; this fourth step is called ribosome recycling. Ribosome recycling occurs after the nascent polypeptide has been released during the termination step. Despite the release of the polypeptide, ribosomes remain bound to the mRNA and tRNA. It is only during the fourth step of translation that ribosomes are ultimately released from the mRNA, split into subunits, and are free to bind new mRNA, thus the term "ribosome recycling." This step is essential to the viability of cells. In bacteria, it is catalyzed by two proteins, elongation factor G and ribosome recycling factor, a near perfect structural mimic of tRNA. Eukaryotic organelles such as mitochondria and chloroplasts possess ribosome recycling factor and elongation factor G homologues, but the nature of ribosome recycling in eukaryotic cytoplasm is still under investigation. In this review, the discovery of ribosome recycling and the basic mechanisms involved are discussed so that textbook writers and teachers can include this vital step, which is just as important as the three conventional steps, in sections dealing with protein synthesis.

  16. 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


    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.

  17. A cell-free expression and purification process for rapid production of protein biologics. (United States)

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


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

  18. Recombinant Protein Production and Insect Cell Culture and Process (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)


    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.

  19. Characteristics and concepts of dynamic hub proteins in DNA processing machinery from studies of RPA. (United States)

    Sugitani, Norie; Chazin, Walter J


    DNA replication, damage response and repair require the coordinated action of multi-domain proteins operating within dynamic multi-protein machines that act upon the DNA substrate. These modular proteins contain flexible linkers of various lengths, which enable changes in the spatial distribution of the globular domains (architecture) that harbor their essential biochemical functions. This mobile architecture is uniquely suited to follow the evolving substrate landscape present over the course of the specific process performed by the multi-protein machinery. A fundamental advance in understanding of protein machinery is the realization of the pervasive role of dynamics. Not only is the machine undergoing dynamic transformations, but the proteins themselves are flexible and constantly adapting to the progression through the steps of the overall process. Within this dynamic context the activity of the constituent proteins must be coordinated, a role typically played by hub proteins. A number of important characteristics of modular proteins and concepts about the operation of dynamic machinery have been discerned. These provide the underlying basis for the action of the machinery that reads DNA, and responds to and repairs DNA damage. Here, we introduce a number of key characteristics and concepts, including the modularity of the proteins, linkage of weak binding sites, direct competition between sites, and allostery, using the well recognized hub protein replication protein A (RPA).

  20. 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


    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...

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

    Coura Oliveira, Tatiana; Lopes Lima, Samuel; Bressan, Josefina


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

  2. Finding the "bio" in biobased products: electrophoretic identification of wheat proteins in processed products. (United States)

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


    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

  3. Protein oxidation in processed cheese slices treated with pulsed light technology. (United States)

    Fernández, M; Ganan, M; Guerra, C; Hierro, E


    The effect of pulsed light technology on protein oxidation was studied in sliced processed cheese by measuring the protein-bound carbonyls with a spectrophotometric DNPH assay. Bovine serum albumin was also tested as a protein standard. Fluences of 0.7, 2.1, 4.2, 8.4 and 11.9 J/cm(2) were applied to vacuum-packaged cheese slices and to an aqueous solution of the protein. Treatments up to 4.2 J/cm(2) did not promote protein oxidation immediately after flashing either in cheese or in the standard. Samples treated with 8.4 and 11.9 J/cm(2) showed significantly higher carbonyl amounts than non-treated ones. Protein oxidation increased along cheese storage at 4°C, and differences among treatments remained. Further studies on the sensory properties will be needed to clarify the impact of pulsed light on processed cheese quality.

  4. Effect of high hydrostatic pressure processing on in vitro digestion of milk proteins and fats (United States)

    The use of high hydrostatic pressure processing (HPP) is increasing in popularity in the food industry. Its ability to modify milk proteins and fats suggests that it may be useful in creating foods that suppress appetite; however, its effect on the digestibility of proteins and fats is unclear. The...

  5. 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


    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 dimethy

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

  7. Design strategies for integrated protein purification processes: challenges, progress and outlook

    NARCIS (Netherlands)

    Nfor, B.; Ahamed, T.; Dedem, G.; Wielen, van der L.; Sandt, van de E.; Eppink, M.H.M.; Ottens, M.


    The key to successful and efficient protein purification is the selection of the most appropriate purification techniques and their combination in a logical way to obtain the desired purification in the minimum number of steps. However, the rationalization of protein purification process development

  8. Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process (United States)

    Mirsadeghi, Somayeh; Dinarvand, Rassoul; Ghahremani, Mohammad Hossein; Hormozi-Nezhad, Mohammad Reza; Mahmoudi, Zohreh; Hajipour, Mohammad Javad; Atyabi, Fatemeh; Ghavami, Mahdi; Mahmoudi, Morteza


    Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called ``protein corona'') upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) ``see'' the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)).Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades

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



    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...

  10. Processing Pisum sativum seed storage protein precursors in vitro

    Institute of Scientific and Technical Information of China (English)



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

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

    Directory of Open Access Journals (Sweden)

    Akmal Yuldashev


    Full Text Available Electron microscopic and immunоfluorescent study in rats aged 1 and 3 days after birth allowed to establish a process of absorption of protein from the small intestine into the lymph and blood. Blood homeostasis was provided by the proteins filtrated from glomerular capillaries of nephrons and reabsorbed by the epithelial cells in canaliculi of nephrons. The absorbed natural heterologous protein was depleted by lysosomes of epithelial cells of intestine and kidneys and macrophages. It supported not only blood homeostasis but also prevented loss of protein by an organism, formed sites for its digestion in the organism.

  12. Extraction and purification methods in downstream processing of plant-based recombinant proteins. (United States)

    Łojewska, Ewelina; Kowalczyk, Tomasz; Olejniczak, Szymon; Sakowicz, Tomasz


    During the last two decades, the production of recombinant proteins in plant systems has been receiving increased attention. Currently, proteins are considered as the most important biopharmaceuticals. However, high costs and problems with scaling up the purification and isolation processes make the production of plant-based recombinant proteins a challenging task. This paper presents a summary of the information regarding the downstream processing in plant systems and provides a comprehensible overview of its key steps, such as extraction and purification. To highlight the recent progress, mainly new developments in the downstream technology have been chosen. Furthermore, besides most popular techniques, alternative methods have been described.

  13. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. (United States)

    Münch, Christian; Harper, J Wade


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

  14. Downregulation of the microtubule associated protein tau impairs process outgrowth and myelin basic protein mRNA transport in oligodendrocytes. (United States)

    Seiberlich, Veronika; Bauer, Nina G; Schwarz, Lisa; Ffrench-Constant, Charles; Goldbaum, Olaf; Richter-Landsberg, Christiane


    Oligodendrocytes, the myelin forming cells of the CNS, are characterized by their numerous membranous extensions, which enwrap neuronal axons and form myelin sheaths. During differentiation oligodendrocytes pass different morphological stages, downregulate the expression of the proteoglycan NG2, and acquire major myelin specific proteins, such as myelin basic proteins (MBP) and proteolipid protein. MBP mRNA is transported in RNA granules along the microtubules (MTs) to the periphery and translated locally. MTs participate in the elaboration and stabilization of the myelin forming extensions and are essential for cellular sorting processes. Their dynamic properties are regulated by microtubule associated proteins (MAPs). The MAP tau is present in oligodendrocytes and involved in the regulation and stabilization of the MT network. To further elucidate the functional significance of tau in oligodendrocytes, we have downregulated tau by siRNA technology and studied the effects on cell differentiation and neuron-glia contact formation. The data show that tau knockdown impairs process outgrowth and leads to a decrease in MBP expression. Furthermore, MBP mRNA transport to distant cellular extensions is impaired and cells remain in the NG2 stage. In myelinating cocultures with dorsal root ganglion neurons, oligodendrocyte precursor cells after tau miR RNA lentiviral knockdown develop into NG2 positive cells with very long and thin processes, contacting axons loosely, but fail to form internodes. This demonstrates that tau is important for MBP mRNA transport and involved in process formation. The disturbance of the balance of tau leads to abnormalities in oligodendrocyte differentiation, neuron-glia contact formation and the early myelination process.

  15. Effect of radiation processing on antinutrients, in-vitro protein digestibility and protein efficiency ratio bioassay of legume seeds (United States)

    El-Niely, Hania F. G.


    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 ( pphytic 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.

  16. 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. (United States)

    Nuez, B; Rojo, F; Salas, M


    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.

  17. 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


    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...... proteins involved in neuronal process extension and maintenance are both more heavily modified and more frequently regulated at a PTM level. This suggests a clear role not only for PTMs in these processes, but possibly also for heavy protein modification in general. BIOLOGICAL SIGNIFICANCE: This study...... 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...

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

    Institute of Scientific and Technical Information of China (English)

    GAO Minjie; SHI Zhongping


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

  19. 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.


    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)

  20. Model-based Rational and Systematic Protein Purification Process Development: A Knowledge-based Approach

    NARCIS (Netherlands)

    Kungah Nfor, B.


    The increasing market and regulatory (quality and safety) demands on therapeutic proteins calls for radical improvement in their manufacturing processes. Addressing these challenges requires the adoption of strategies and tools that enable faster and more efficient process development. This thesis i

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

    DEFF Research Database (Denmark)

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


    A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system...

  2. Biochemistry in an Industrial Context: Methods of Protein Purification and Downstream Processing. (United States)

    Weathers, Pamela J.


    Explores a graduate level bioprocess engineering course in protein purification and downstream processing. Designed to provide students with hands-on training in the design and implementation of product processing for the biotechnology industry. Includes syllabus and plan of study. (MVL)

  3. Using Simple Manipulatives to Improve Student Comprehension of a Complex Biological Process: Protein Synthesis (United States)

    Guzman, Karen; Bartlett, John


    Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article…

  4. [Biological Role of Oligomerny Matriksny of Protein of the Cartilage in Exchange Processes Connecting Tissue]. (United States)

    Belova, Yu S


    In the review the literary data on studying of biological role of a oligomerny matriksny of protein of the cartilage in exchange processes connecting tissue at people and animals are provided, and also results of own researches on definition of a oligomerny matriksny of protein of the cartilage as a modern marker of a metabolism of an articulate cartilage at children from undifferentiated displaziy conjunctive tissue are briefly described.

  5. An improved strategy for easy process monitoring and advanced purification of recombinant proteins. (United States)

    Miladi, Baligh; Dridi, Cyrine; El Marjou, Ahmed; Boeuf, Guilhem; Bouallagui, Hassib; Dufour, Florence; Di Martino, Patrick; Elm'selmi, Abdellatif


    In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.

  6. 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


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

  7. Degradation of LIM domain-binding protein three during processing of Spanish dry-cured ham. (United States)

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


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

  8. Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes

    Directory of Open Access Journals (Sweden)

    Diaz Celine


    Full Text Available Abstract Background 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets. Results In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds. Conclusion Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.

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



    Extensive proteolysis takes place during the processing of dry-cured ham due to the action of muscle peptidases. The aim of this work was to study the degradation of LIM domain binding protein 3 (LDB3), which is located at the Z-lines of the sarcomere, at different times during the Spanish dry-cured ham processing (2, 3.5, 5, 6.5, and 9 months). A total of 107 peptides have been identified by mass spectrometry, most of them generated from the first region of the protein sequence (position 1-9...

  10. Global proteomic screening of protein allergens and advanced glycation endproducts in thermally processed peanuts. (United States)

    Hebling, Christine M; McFarland, Melinda A; Callahan, John H; Ross, Mark M


    Peanuts (Arachis hypogaea) are the cause of one of the most prevalent food allergies worldwide. Thermal processing (e.g., roasting) of peanuts and peanut-containing foods results in complex chemical reactions that alter structural conformations of peanut proteins, preventing accurate detection of allergens by most immunochemical and targeted screening methodologies. To improve food allergen detection and support more accurate food labeling, traditional methods for peanut protein extraction were modified to include protein denaturants and solubilization agents. Qualitative characterization by SDS-PAGE and Western blot analyses of raw and variably roasted peanut extracts confirmed improvements in total protein recovery and provided evidence for the incorporation of Ara h 1, Ara h 3, and, to a lesser extent, Ara h 2 into high molecular weight protein complexes upon roasting. Relative quantification of allergens in peanut lysates was accomplished by label-free spectral feature (MS1) LC-MS/MS methodologies, by which peanut allergen peptides exhibiting a differential MS response in raw versus roasted peanuts were considered to be candidate targets of thermal modification. Identification of lysine-modified Maillard advanced glycation endproducts (AGE) by LC-MS/MS confirmed the formation of (carboxymethyl)lysine (CML), (carboxyethyl)lysine (CEL), and pyrraline (Pyr) protein modifications on Ara h 1 and Ara h 3 tryptic peptides in roasted peanut varieties. These results suggest that complex processed food matrices require initial analysis by an untargeted LC-MS/MS approach to determine optimum analytes for subsequent targeted allergen analyses.


    Directory of Open Access Journals (Sweden)



    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.

  12. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography. (United States)

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


    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.

  13. 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:


    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.

  14. Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity. (United States)

    Turunen, S; Käpylä, E; Terzaki, K; Viitanen, J; Fotakis, C; Kellomäki, M; Farsari, M


    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.

  15. 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: [VTT Technical Research Centre of Finland, PO Box 1300, 33101 Tampere (Finland)


    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.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  17. Antioxidant protection of proteins and lipids in processed pork loin chops through feed supplementation with avocado. (United States)

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


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

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

    NARCIS (Netherlands)

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


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

  19. Determination of processed animal proteins, including meat and bone meal, in animal feed

    NARCIS (Netherlands)

    Gizzi, G.; Holst, von C.; Baeten, V.; Berben, G.; Raamsdonk, van L.W.D.


    The presence of processed animal proteins (PAP), including meat and bone meal (MBM) from various species, in animal feed was investigated. It was demonstrated that microscopy is the most reliable method for enforcing the current total MBM ban in the European Uion (EU). It was shown that near infrare

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

    Directory of Open Access Journals (Sweden)

    Yiwen Xiang

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

  1. High-Pressure-High-Temperature Processing Reduces Maillard Reaction and Viscosity in Whey Protein-Sugar Solutions. (United States)

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


    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.

  2. Protein (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...


    Sawant, Akshada; Kothandapani, Anbarasi; Zhitkovich, Anatoly; Sobol, Robert W.; Patrick, Steve M.


    Mismatch repair (MMR) deficiency gives rise to cisplatin resistance and can lead to poor prognosis in cancers. Various models have been proposed to explain this low level of resistance caused due to loss of MMR proteins. We have shown that MMR proteins are required to maintain cisplatin interstrand cross-links (ICLs) on the DNA leading to increased cellular sensitivity. In our previous studies, we have shown that BER processing of the cisplatin ICLs is mutagenic. Polymerase β (Polβ) can generate mismatches which leads to the activation and the recruitment of mismatch repair proteins. In this paper, we distinguished between the requirement of different downstream MMR proteins for maintaining cisplatin sensitivity. We show that the MutSα (MSH2-MSH6) heterocomplex is required to maintain cisplatin sensitivity, whereas the Mutsβ complex has no effect. These results can be correlated with the increased repair of cisplatin ICLs and ICL induced DNA double strand breaks (DSBs) in the resistant cells. Moreover, we show that MLH1 proficient cells displayed a cisplatin sensitive phenotype when compared with the MLH1 deficient cells and the ATPase activity of MLH1 is essential to mediate this effect. Based on these results, we propose that MutSα as well as the downstream MMR pathway proteins are essential to maintain a cisplatin sensitive phenotype as a consequence of processing Polβ induced mismatches at sites flanking cisplatin ICLs. PMID:26519826

  4. Integration of protein processing steps on a droplet microfluidics platform for MALDI-MS analysis. (United States)

    Chatterjee, Debalina; Ytterberg, A Jimmy; Son, Sang Uk; Loo, Joseph A; Garrell, Robin L


    A droplet-based (digital) microfluidics platform has been developed to prepare and purify protein samples for measurement by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Liquid droplets are moved in air by sequentially applying an electric potential to an array of electrodes patterned beneath a hydrophobic dielectric layer. We show that a complete integrated sequence of protein processing steps can be performed on this platform, including disulfide reduction, alkylation, and enzymatic digestion, followed by cocrystallization with a MALDI matrix and analysis of the sample in situ by MALDI-MS. Proteins carbonic anhydrase, cytochrome c, and ubiquitin were used to demonstrate the digestion and postdigestion steps; insulin, serum albumin, and lysozyme were used to illustrate the complete sequence of protein processing steps available with the platform. Several functional improvements in the platform are reported, notably, the incorporation of acetonitrile in the protein droplets to facilitate movement, and patterning the device surfaces to optimize sample crystallization. The method is fast, simple, repeatable, and results in lower reagent consumption and sample loss than conventional techniques for proteomics sample preparation.

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

    Directory of Open Access Journals (Sweden)

    Sahar Golaghaiee


    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.

  6. Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study. (United States)

    Sakai, Shinobu; Adachi, Reiko; Akiyama, Hiroshi; Teshima, Reiko; Doi, Hirotoshi; Shibata, Haruki


    Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

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

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    Hamed Safafar


    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.

  8. Lipase-catalyzed process for biodiesel production: protein engineering and lipase production. (United States)

    Hwang, Hyun Tae; Qi, Feng; Yuan, Chongli; Zhao, Xuebing; Ramkrishna, Doraiswami; Liu, Dehua; Varma, Arvind


    Biodiesel is an environment-friendly and renewable fuel produced by transesterification of various feedstocks. Although the lipase-catalyzed biodiesel production has many advantages over the conventional alkali catalyzed process, its industrial applications have been limited by high-cost and low-stability of lipase enzymes. This review provides a general overview of the recent advances in lipase engineering, including both protein modification and production. Recent advances in biotechnology such as in protein engineering, recombinant methods and metabolic engineering have been employed but are yet to impact lipase engineering for cost-effective production of biodiesel. A summary of the current challenges and perspectives for potential solutions are also provided.

  9. Reproducible radiation-damage processes in proteins irradiated by intense x-ray pulses (United States)

    Hau-Riege, Stefan P.; Bennion, Brian J.


    X-ray free-electron lasers have enabled femtosecond protein nanocrystallography, a novel method to determine the structure of proteins. It allows time-resolved imaging of nanocrystals that are too small for conventional crystallography. The short pulse duration helps in overcoming the detrimental effects of radiation damage because x rays are scattered before the sample has been significantly altered. It has been suggested that, fortuitously, the diffraction process self-terminates abruptly once radiation damage destroys the crystalline order. Our calculations show that high-intensity x-ray pulses indeed trigger a cascade of damage processes in ferredoxin crystals, a particular metalloprotein of interest. However, we found that the damage process is initially not completely random. Correlations exist among the protein monomers, so that Bragg diffraction still occurs in the damaged crystals, despite significant atomic displacements. Our results show that the damage process is reproducible to a certain degree, which is potentially beneficial for the orientation step in single-molecule imaging.

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

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol


    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.

  11. Interlaboratory Study of ELISA Kits for the Detection of Egg and Milk Protein in Processed Foods. (United States)

    Kato, Shigeki; Yagi, Takahiro; Kato, Ayako; Yamamoto, Shunsuke; Akimoto, Masanobu; Arihara, Keizo


    The labeling of seven specific allergenic ingredients (egg, milk, wheat, buckwheat, peanut, shrimp, and crab) is mandatory in Japan. To ensure proper labeling, two kinds of ELISA kits using polyclonal antibodies have been developed. However, we developed two novel ELISA kits using monoclonal antibodies with improved specificity, the Allergeneye ELISA Egg (AE-Egg) and Allergeneye ELISA Milk (AE-Milk) Kits, to detect egg and milk proteins in processed foods, respectively. Five types of processed food containing 10 mg/kg of egg or milk soluble protein were prepared for an interlaboratory study of the performance of these kits. The kits showed a relatively high reproducibility level of interlaboratory precision (AE-Egg RSDR, 3.7-5.7%; AE-Milk RSDR, 6.8-10.5%) and satisfied the recovery rate stipulated by Japanese guidelines (AE-Egg, 61.6-89.3%; AE-Milk, 52.1-67%) for all processed foods. Our results suggest that the AE-Egg and AE-Milk Kits are precise and reliable tools for detecting egg or milk proteins in processed foods.

  12. Sycamore amyloplasts can import and process precursors of nuclear encoded chloroplast proteins. (United States)

    Strzalka, K; Ngernprasirtsiri, J; Watanabe, A; Akazawa, T


    Amyloplasts isolated from white-wild suspension-cultured cells of sycamore (Acer pseudoplatanus L.) are found to import and process the precursor of the small subunit (pS) of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach, but they lack the ability to form its holoenzyme due to the absence of both the large subunit and its binding-protein. They also import the precursor of the 33-kDa extrinsic protein (p33-kDa) of the O2-evolving complex of Photosystem II from spinach, but process is only to an intermediate form (i33-kDa). Chloroplasts from green-mutant cells of sycamore process p33-kDa to its mature form in this heterologous system. These results suggest that the thylakoid-associated protease responsible for the second processing step of p33-kDa is missing in amyloplasts, possibly due to the absence of thylakoid-membranes. In contrast, the apparent import of the precursor of the light-harvesting chlorophyll a/b-binding apoprotein (pLHCP) from spinach was not detected. Sycamore amyloplasts may lack the ability to import this particular thylakoid-protein, or rapidly degrade the imported molecules in the absence of thylakoid-membranes for their proper insertion.

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

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    Andrew McAloon


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

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

    DEFF Research Database (Denmark)

    Belsham, Graham

    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...... or with FMDV 3Cpro using a “single cycle” alphavirus vector based on Semliki Forest virus (SFV). Cattle vaccinated with these rSFV-FMDV vectors alone, produced anti-FMDV antibodies but the immune response was insufficient to give protection against FMDV challenge. However, vaccination with these vectors primed...... a much stronger immune response against FMDV post-challenge. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector...

  15. High pressure processing of meat: effects on ultrastructure and protein digestibility. (United States)

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


    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.

  16. Scalable organic solvent free supercritical fluid spray drying process for producing dry protein formulations. (United States)

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


    In this study, we evaluated the influence of supercritical carbon dioxide (scCO2) spray drying conditions, in the absence of organic solvent, on the ability to produce dry protein/trehalose formulations at 1:10 and 1:4 (w/w) ratios. When using a 4L drying vessel, we found that decreasing the solution flow rate and solution volume, or increasing the scCO2 flow rate resulted in a significant reduction in the residual water content in dried products (Karl Fischer titration). The best conditions were then used to evaluate the ability to scale the scCO2 spray drying process from 4L to 10L chamber. The ratio of scCO2 and solution flow rate was kept constant. The products on both scales exhibited similar residual moisture contents, particle morphologies (SEM), and glass transition temperatures (DSC). After reconstitution, the lysozyme activity (enzymatic assay) and structure (circular dichroism, HP-SEC) were fully preserved, but the sub-visible particle content was slightly increased (flow imaging microscopy, nanoparticle tracking analysis). Furthermore, the drying condition was applicable to other proteins resulting in products of similar quality as the lysozyme formulations. In conclusion, we established scCO2 spray drying processing conditions for protein formulations without an organic solvent that holds promise for the industrial production of dry protein formulations.

  17. 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.

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

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    Jenna M. Ramaker


    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

  19. Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies. (United States)

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


    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.

  20. Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process. (United States)

    Trezzani, I; Nadri, M; Dorel, C; Lejeune, P; Bellalou, J; Lieto, J; Hammouri, H; Longin, R; Dhurjati, P


    On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.

  1. Pri sORF peptides induce selective proteasome-mediated protein processing. (United States)

    Zanet, J; Benrabah, E; Li, T; Pélissier-Monier, A; Chanut-Delalande, H; Ronsin, B; Bellen, H J; Payre, F; Plaza, S


    A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

  2. Low-Temperature Processable Block Copolymers That Preserve the Function of Blended Proteins. (United States)

    Iwasaki, Yasuhiko; Takemoto, Kyohei; Tanaka, Shinya; Taniguchi, Ikuo


    Low-temperature processable polymers have attracted increasing interest as ecological materials because of their reduced energy consumption during processing and suitability for making composites with heat-sensitive biomolecules at ambient temperature. In the current study, low-temperature processable biodegradable block copolymers were synthesized by ring-opening polymerization of l-lactide (LLA) using polyphosphoester as a macroinitiator. The polymer films could be processed under a hydraulic pressure of 35 MPa. The block copolymer films swelled in water because the polyphosphoester block was partially hydrated. Interestingly, the swelling ratio of the films changed with temperature. The pressure-induced order-to-disorder transition of the block copolymers was characterized by small-angle X-ray scattering; a crystallinity reduction in the block copolymers was observed after application of pressure. The crystallinity of the block copolymers was recovered after removing the applied pressure. The Young's modulus of the block copolymer films increased as the LLA unit content increased. Moreover, the modulus did not change after multiple processing cycles and the recyclability of the block copolymers was also confirmed. Finally, polymer films with embedded proteinase K as a model protein were prepared. The activity of catalase loaded into the polymer films was evaluated after processing at different temperatures. The activity of catalase was preserved when the polymer films were processed at room temperature but was significantly reduced after high-temperature processing. The suitability of low-temperature processable biodegradable polymers for making biofunctional composites without reducing protein activity was clarified. These materials will be useful for biomedical and therapeutic applications.


    Directory of Open Access Journals (Sweden)

    T. I. Romanyuk


    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.

  4. Circular dichroism and infrared spectroscopic characterization of secondary structure components of protein Z during mashing and boiling processes. (United States)

    Han, Yupeng; Wang, Jinjing; Li, Yongxian; Hang, Yu; Yin, Xiangsheng; Li, Qi


    In beer brewing, protein Z is hypothesized to stabilize beer foam. However, few investigations have revealed the relationship between conformational alterations to protein Z during the brewing process and beer foam. In this report, protein Z from sweet wort was isolated during mashing and boiling processes. Circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) were used to monitor the structural characteristics of protein Z. The results showed that the α-helix and β-sheet content decreased, whereas the content of β-turn and random coil increased. The complex environment rich in polysaccharides may facilitate conformational alterations and modifications to protein Z. Additionally, the formation of extended structural features to protein Z provides access to reactive amino acid side chains that can undergo modifications and the exposure of hydrophobic core regions of the protein. Analyzing structural transformations should provide a deeper understanding of the mechanism of protein Z on maintaining beer foam.

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

    Directory of Open Access Journals (Sweden)

    Shun Mao


    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.

  6. Near-infrared reflectance spectroscopy for predicting amino acids content in intact processed animal proteins. (United States)

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


    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 < 3.0%) obtained for the prediction of amino acids in intact processed animal proteins opens an enormous expectative for the on-line implementation of NIRS technology in the processing and marketing of these important protein feed ingredients, alleviating the costs and time associated with the routine quality controls.

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

    Indian Academy of Sciences (India)

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


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

  8. Molecular Characterization of Soybean Mosaic Virus NIa Protein and its Processing Event in Bacterial Expression

    Directory of Open Access Journals (Sweden)

    Bong K. Choi


    Full Text Available Soybean mosaic virus (SMV-CN18 is an Rsv resistance-breaking (RB isolate to overcome soybean resistance genes Rsv1, Rsv3 and Rsv4. The aim of this study was to characterize nuclear inclusion protein a (NIa protein of RB isolate at the molecular level and demonstrate its processing into genome-linked protein (VPg and NIa-Pro domains in Esherichia coli containing a bacterial expression pET vector inserted with NIa gene. The full-length of NIa gene was synthesized by reverse transcription-polymerase chain reaction (RT-PCR and its 1298 nucleotides (nt and 432 amino acids (aa were deduced. The nt and aa sequences of NIa gene of SMV-CN18 shared high identities with the corresponding sequences of the NIa gene of the known SMV isolates, suggesting that the NIa is a highly conserved protein. The NIa-Pro domain contains a highly conserved structural motif for proteolysis, while the VPg domain contains a nuclear localization signal (NLS, a putative NTP-binding site and cellular factor-binding sites. The phylogenetic tree revealed that less divergence of NIa protein exists among twelve SMV isolates, which can be supported by a low bootstrap value between clades. In addition, the full-length of NIa gene, amplified by RT-PCR, was ligated into pET-28b E. coli expression vector with an N-terminal His6-tag. Optimal conditions for expression were at 1mM treatment of IPTG at 25°C for 5 hr. The released protein from bacterial lysates remained soluble and proved the processing form of the NIa polyprotein. E. coli expression system shows the processed product of 29 kDa VPg in SDS-PAGE confirmed by western blot analysis in both crude extracts and purified elution products, using Ni2+-NTA resin. The present study indicates that the N-terminal region of NIa which is processed and expressed in bacteria.

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

    DEFF Research Database (Denmark)

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


    -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...

  10. Construction of a Cell Based Sensor for the Detection of Autoinducer-2 (Reprint) (United States)


    Zhu J, Miller M.B., Vance R.E., Dziejman M, Bassler B.L., “Quorum-sensing regulators control virulence gene expression in Vibrio cholerae ,” Proc...The assay utilizes the reporter strain Vibrio harveyi BB170, a mutant strain of V. harveyi which does not produce AI-2, which produces...expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway,” Mol. Microbiol. 13: 273–286 (1994). [6

  11. Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation


    Zhu, Jie; Hixon, Mark S.; Globisch, Daniel; Kaufmann, Gunnar F.; Janda, Kim D.


    In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2 related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet t...

  12. Comparison of batch and continuous multi-column protein A capture processes by optimal design. (United States)

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


    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.

  13. Phylogenetic Gaussian process model for the inference of functionally important regions in protein tertiary structures. (United States)

    Huang, Yi-Fei; Golding, G Brian


    A critical question in biology is the identification of functionally important amino acid sites in proteins. Because functionally important sites are under stronger purifying selection, site-specific substitution rates tend to be lower than usual at these sites. A large number of phylogenetic models have been developed to estimate site-specific substitution rates in proteins and the extraordinarily low substitution rates have been used as evidence of function. Most of the existing tools, e.g. Rate4Site, assume that site-specific substitution rates are independent across sites. However, site-specific substitution rates may be strongly correlated in the protein tertiary structure, since functionally important sites tend to be clustered together to form functional patches. We have developed a new model, GP4Rate, which incorporates the Gaussian process model with the standard phylogenetic model to identify slowly evolved regions in protein tertiary structures. GP4Rate uses the Gaussian process to define a nonparametric prior distribution of site-specific substitution rates, which naturally captures the spatial correlation of substitution rates. Simulations suggest that GP4Rate can potentially estimate site-specific substitution rates with a much higher accuracy than Rate4Site and tends to report slowly evolved regions rather than individual sites. In addition, GP4Rate can estimate the strength of the spatial correlation of substitution rates from the data. By applying GP4Rate to a set of mammalian B7-1 genes, we found a highly conserved region which coincides with experimental evidence. GP4Rate may be a useful tool for the in silico prediction of functionally important regions in the proteins with known structures.

  14. Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution. (United States)

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


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

  15. High intakes of protein and processed meat associate with increased incidence of type 2 diabetes. (United States)

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


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

  16. Effects of phytase-assisted processing method on physicochemical and functional properties of soy protein isolate. (United States)

    Wang, Hongjian; Chen, Yeming; Hua, Yufei; Kong, Xiangzhen; Zhang, Caimeng


    Phytate is an important antinutritional factor in food products. In this study, a phytase-assisted processing method was used to produce low-phytate soybean protein isolate (SPI) samples, and their physicochemical and functional properties were examined. Hydrolysis condition at low temperature (room temperature) and pH 5.0 was better than that recommended by manufacturer (pH 5.0, 55 °C) at keeping the properties of SPI, so the former condition was selected to prepare SPI samples with phytate contents of 19.86-0.11 mg/g by prolonging hydrolysis time (0 (traditional method), 5, 10, 20, 40, and 60 min). Ash content (R(2) = 0.940), solubility (R(2) = 0.983), ζ-potential value (R(2) = 0.793), denaturation temperatures (β-conglycinin, R(2) = 0.941; glycinin, R(2) = 0.977), emulsifying activity index (R(2) = 0.983), foaming capacity (R(2) = 0.955), and trypsin inhibitor activity (R(2) = 0.821) of SPI were positively correlated with phytate content, whereas protein content (R(2) = 0.876), protein recovery (R(2) = 0.781), emulsifying stability index (R(2) = 0.953), foaming stability (R(2) = 0.919), gel hardness (R(2) = 0.893), and in vitro digestibility (R(2) = 0.969) were negatively correlated with phytate content. Simulated gastrointestinal digestion and subsequent dialysis showed that percentages of dialyzable Zn and Ca were increased with decreasing phytate content, whereas the amounts of dialyzable Zn and Ca revealed different behaviors: the former was increased and the latter was decreased. Circular dichroism spectra showed that secondary structure of SPI was changed by phytase. Compared with traditional processing method, the phytase-assisted processing method could produce SPI with lower phytate and higher protein contents, which had better in vitro digestibility and could be used to prepare gels with higher hardness by partially losing some other functional properties.

  17. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Eran Bosis

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

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

    Directory of Open Access Journals (Sweden)

    Dustin E Bosch

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

  20. Exact protein distributions for stochastic models of gene expression using partitioning of Poisson processes (United States)

    Pendar, Hodjat; Platini, Thierry; Kulkarni, Rahul V.


    Stochasticity in gene expression gives rise to fluctuations in protein levels across a population of genetically identical cells. Such fluctuations can lead to phenotypic variation in clonal populations; hence, there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. Here, we invoke the partitioning property of Poisson processes to develop a mapping that significantly simplifies the analysis of stochastic models of gene expression. The mapping leads to exact protein distributions using results for mRNA distributions in models with promoter-based regulation. Using this approach, we derive exact analytical results for steady-state and time-dependent distributions for the basic two-stage model of gene expression. Furthermore, we show how the mapping leads to exact protein distributions for extensions of the basic model that include the effects of posttranscriptional and posttranslational regulation. The approach developed in this work is widely applicable and can contribute to a quantitative understanding of stochasticity in gene expression and its regulation.

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

    Directory of Open Access Journals (Sweden)

    Pang Bin


    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.

  2. The photodamage process of pigments and proteins of PSI complexes from Spinacia Oleracea L.

    Institute of Scientific and Technical Information of China (English)


    Purified PSI complexes from Spinacia Oleracea L. were exposed to the strong light (PFD=2300 μmol m-2s-1)for various period. Along with the illumination the photodamage process of pigments and proteins of PSI complexes was investigated using absorption, fluorescence, circular dichroism (CD) spectroscopy and SDS.PAGE. It was found from the optical absorption spectra that the maximal absorbance of PSI complexes decreased and maximal peaks blue-shifted during the illumination, and the forth derivative spectra demonstrated that the absorbance decreasing at red region mainly resulted from the aborbance decreasing of the long wavelength Chla, implying that the long.wavelength Chla was readily to be bleached. The CD signals contributed by LHCI decreased more rapidly than other CD signals contributed by Chla and Carotenoid, indicating that the LHCI was more sensitive to light than core complexes. It was observed by SDS-PAGE that some small polypeptides of PSI complexes were damaged earlier than reaction center proteins PsaA and PsaB. Lhca3, Lhca2 and PsaD were the early degraded proteins during illumination. In addition, it is also observed that the insoluble-cohesive-denatured proteins appeared after prolonged illumination.

  3. Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct. (United States)

    Berger, Christian; Berndt, Sandra; Pichert, Annelie; Theisgen, Stephan; Huster, Daniel


    A protocol for the efficient isotopic labeling of large G protein-coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L-tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell-cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell-cell communication by the addition of indole during expression. Discrete concentrations of indole and (15) N2 -L-tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ∼15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium.

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

    Directory of Open Access Journals (Sweden)

    Xihong Zhao


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

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

    DEFF Research Database (Denmark)

    Schou, Jørgen; Matei, Andreea; Constantinescu, Catalin


    the ejection and deposition of lysozyme. This can be called an “inverse MAPLE” process, since the ratio of “matrix” to film material in the target is 10:90, which is inverse of the typical MAPLE process where the film material is dissolved in the matrix down to several wt.%. Lysozyme is a well-known protein...... 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...

  6. The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2. (United States)

    Beckmann, Anna-Madeleine; Glebov, Konstantin; Walter, Jochen; Merkel, Olaf; Mangold, Martin; Schmidt, Frederike; Becker-Pauly, Christoph; Gütschow, Michael; Stirnberg, Marit


    Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ.

  7. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation. (United States)

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe


    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

  8. Influence of thermal processing on IgE reactivity to lentil and chickpea proteins. (United States)

    Cuadrado, Carmen; Cabanillas, Beatriz; Pedrosa, Mercedes M; Varela, Alejandro; Guillamón, Eva; Muzquiz, Mercedes; Crespo, Jesús F; Rodriguez, Julia; Burbano, Carmen


    In the last years, legume proteins are gaining importance as food ingredients because of their nutraceutical properties. However, legumes are also considered relevant in the development of food allergies through ingestion. Peanuts and soybeans are important food allergens in Western countries, while lentil and chickpea allergy are more relevant in the Mediterranean area. Information about the effects of thermal-processing procedures at various temperatures and conditions is scarce; therefore, the effect of these procedures on legume allergenic properties is not defined so far. The SDS-PAGE and IgE-immunoblotting patterns of chickpeas and lentils were analyzed before and after boiling (up to 60 min) and autoclaving (1.2 and 2.6 atm, up to 30 min). The results indicated that some of these treatments reduce IgE binding to lentil and chickpea, the most important being harsh autoclaving. However, several extremely resistant immunoreactive proteins still remained in these legumes even after this extreme treatment.

  9. Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin. (United States)

    Axmann, Sonja; Adler, Andreas; Brandstettner, Agnes Josephine; Spadinger, Gabriela; Weiss, Roland; Strnad, Irmengard


    Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of 'false-positive' PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR.

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

    Directory of Open Access Journals (Sweden)

    Philipp A Jaeger

    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.

  11. Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species. (United States)

    Caccia, Silvia; Chakroun, Maissa; Vinokurov, Konstantin; Ferré, Juan


    Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolytic activation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein.

  12. 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)


    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

  13. Cajal body proteins differentially affect the processing of box C/D scaRNPs. (United States)

    Enwerem, Isioma I; Wu, Guowei; Yu, Yi Tao; Hebert, Michael D


    Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.

  14. Cajal body proteins differentially affect the processing of box C/D scaRNPs.

    Directory of Open Access Journals (Sweden)

    Isioma I Enwerem

    Full Text Available Small nuclear ribonucleoproteins (snRNPs, which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs. The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.

  15. 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


    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.

  16. Nixtamalized flour from quality protein maize (Zea mays L). optimization of alkaline processing. (United States)

    Milán-Carrillo, J; Gutiérrez-Dorado, R; Cuevas-Rodríguez, E O; Garzón-Tiznado, J A; Reyes-Moreno, C


    Quality of maize proteins is poor, they are deficient in the essential amino acids lysine and tryptophan. Recently, in Mexico were successfully developed nutritionally improved 26 new hybrids and cultivars called quality protein maize (QPM) which contain greater amounts of lysine and tryptophan. Alkaline cooking of maize with lime (nixtamalization) is the first step for producing several maize products (masa, tortillas, flours, snacks). Processors adjust nixtamalization variables based on experience. The objective of this work was to determine the best combination of nixtamalization process variables for producing nixtamalized maize flour (NMF) from QPM V-537 variety. Nixtamalization conditions were selected from factorial combinations of process variables: nixtamalization time (NT, 20-85 min), lime concentration (LC, 3.3-6.7 g Ca(OH)2/l, in distilled water), and steep time (ST, 8-16 hours). Nixtamalization temperature and ratio of grain to cooking medium were 85 degrees C and 1:3 (w/v), respectively. At the end of each cooking treatment the steeping started for the required time. Steeping was finished by draining the cooking liquor (nejayote). Nixtamal (alkaline-cooked maize kernels) was washed with running tap water. Wet nixtamal was dried (24 hours, 55 degrees C) and milled to pass through 80-US mesh screen to obtain NMF. Response surface methodology (RSM) was applied as optimization technique, over four response variables: In vitro protein digestibility (PD), total color difference (deltaE), water absorption index (WAI), and pH. Predictive models for response variables were developed as a function of process variables. Conventional graphical method was applied to obtain maximum PD, WAI and minimum deltaE, pH. Contour plots of each of the response variables were utilized applying superposition surface methodology, to obtain three contour plots for observation and selection of best combination of NT (31 min), LC (5.4 g Ca(OH)2/l), and ST (8.1 hours) for producing

  17. Posttranslational processing of the Epstein-Barr virus-encoded p63/LMP protein.


    Mann, K P; Thorley-Lawson, D


    In this paper we describe the posttranslational processing of the p63/LMP (latent membrane protein) encoded by Epstein-Barr virus in transformed B cells. Specifically, we show that after synthesis, free LMP disappeared with a half-life of about 0.5 h. This was caused by the association of LMP with an insoluble complex. All detectable LMP in the plasma membrane was insoluble. This interaction was resistant to nondenaturing detergents but readily dissociated with 8 M urea or by boiling in 0.5% ...

  18. Processing of recombinant spider silk proteins into tailor-made materials for biomaterials applications. (United States)

    Schacht, Kristin; Scheibel, Thomas


    Spider silk has extraordinary mechanical properties, is biocompatible and biodegradable, and therefore an ideal material for biomedical applications. However, a drawback for any application is the inhomogeneity of spider silk, as seen for other natural materials, as well as the low availability due to the cannibalism of most spiders. Recently, developed recombinant spider silk proteins ensure constant material properties, as well as scalable production, and further the processing into morphologies other than fibres. Biotechnology enables genetic modification, broadening the range of applications, such as implant coatings, scaffolds for tissue engineering, wound dressing devices as well as drug delivery systems.

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

    DEFF Research Database (Denmark)

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


    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...... 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...

  20. Development and validation of a lateral flow assay for the detection of crustacean protein in processed foods. (United States)

    Koizumi, Daisuke; Shirota, Kazuya; Akita, Ryoko; Oda, Hiroshi; Akiyama, Hiroshi


    We developed and validated a novel lateral flow assay for the detection of crustacean protein in processed foods. This assay had high sensitivity; the visual detection limit for shrimp protein extract was 25μg/L, equivalent to 1μg/g protein in a food sample, and results could be obtained within 20min without sophisticated procedures or expensive equipment. Concordance between our assay and another validated quantitative enzyme-linked immunosorbent assay was 97% for commercially processed foods. This assay is rapid, simple, reliable, and highly correlated with validated enzyme-linked immunosorbent assays and is thus suitable for monitoring of food products, especially in food-processing facilities.

  1. Split End Family RNA Binding Proteins: Novel Tumor Suppressors Coupling Transcriptional Regulation with RNA Processing

    Directory of Open Access Journals (Sweden)

    Hairui Su


    Full Text Available Split End (SPEN family proteins have three members: SPEN, RBM15, and RBM15B. SPEN family proteins contain three conserved RNA recognition motifs on the N-terminal region and an SPOC domain on the C-terminal region. RBM15 is fused to MKL1 in chromosome translocation t (1;22, which causes childhood acute megakaryoblastic leukemia (AMKL. Haploinsufficiency of RBM15 in AMKL indicates that RBM15 is a tumor suppressor. Both SPEN and RBM15 are mutated in a variety of cancer types, implying that they are tumor suppressors. SPEN and RBM15are required for the development of multiple organs including hematopoiesis partly via regulating the NOTCH signaling pathway, as well as the WNT signaling pathway in species ranging from Drosophila to mammals. Besides transcriptional regulation, RBM15 regulates RNA export and RNA splicing. In this review, we summarized data in the literature on how the members in SPEN family regulate gene expression at transcription and RNA processing steps. The crosstalk between epigenetic regulation and RNA metabolism is increasingly appreciated in understanding tumorigenesis. Studying the SPEN family of RNA binding proteins will create new perspectives for cancer therapy.

  2. Unraveling the electron transfer processes of a nanowire protein from Geobacter sulfurreducens. (United States)

    Alves, Mónica N; Fernandes, Ana P; Salgueiro, Carlos A; Paquete, Catarina M


    The extracellular electron transfer metabolism of Geobacter sulfurreducens is sustained by several multiheme c-type cytochromes. One of these is the dodecaheme cytochrome GSU1996 that belongs to a new sub-class of c-type cytochromes. GSU1996 is composed by four similar triheme domains (A–D). The C-terminal half of the molecule encompasses the domains C and D, which are connected by a small linker and the N-terminal half of the protein contains two domains (A and B) that form one structural unit. It was proposed that this protein works as an electrically conductive device in G. sulfurreducens, transferring electrons within the periplasm or to outer-membrane cytochromes. In this work, a novel strategy was applied to characterize in detail the thermodynamic and kinetic properties of the hexaheme fragment CD of GSU1996. This characterization revealed the electron transfer process of GSU1996 for the first time, showing that a heme at the edge of the C-terminal of the protein is thermodynamic and kinetically competent to receive electrons from physiological redox partners. This information contributes towards understanding how this new sub-class of cytochromes functions as nanowires, and also increases the current knowledge of the extracellular electron transfer mechanisms in G. sulfurreducens.

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

    CERN Document Server

    Molin, J P Dal


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

  4. Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level. (United States)

    Bahrami, Soroush Alaghehband; Bakhtiari, Nuredin


    We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1β and α-Klotho. In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20mg/ml) and then administrated (200mg/Kg i.p injection) to mice, twice daily for 7days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy. The data illustrated that UA significantly increased SIRT1 (∼3.5±0.3 folds) and SIRT-6 (∼1.5±0.2 folds) proteins overexpression (Presults showed that UA enhanced α-Klotho (∼3.3±0.3) and PGC-1β (∼2.6±0.2 folds) proteins levels (PUA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1β in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging.

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

    Directory of Open Access Journals (Sweden)

    Zebin Wang


    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.

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

    Directory of Open Access Journals (Sweden)

    Barillot Emmanuel


    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

  7. The role of electrostatic interactions in the process of diffusional encounter and docking of electron transport proteins. (United States)

    Kovalenko, I B; Khrushchev, S S; Fedorov, V A; Riznichenko, G Yu; Rubin, A B


    Electrostatic interaction of plastocyanin and cytochrome f in the process of protein-protein complex formation was investigated by computer simulation methods. It was shown that long-range electrostatic interaction promotes energetically favorable mutual orientation of protein molecules at distances between their cofactors shorter than 5 nm. At distances shorter than 3 nm, these electrostatic interactions lead to a significantly detectable increase in the rate of convergence of the cofactors.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

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

    Directory of Open Access Journals (Sweden)

    Chang-Cai Liu

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

  10. Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins. (United States)

    Asenjo, Ana; Villanueva, Nieves


    The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells.

  11. Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants. (United States)

    Buyel, Johannes F; Fischer, Rainer


    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.

  12. Soy protein recovery in a solvent-free process using continuous liquid-solid circulating fluidized bed ion exchanger. (United States)

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


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

  13. Understanding leaf membrane protein extraction to develop a food-grade process

    NARCIS (Netherlands)

    Tamayo Tenorio, Angelica; Boom, Remko M.; Goot, van der Atze Jan


    Leaf membrane proteins are an underutilised protein fraction for food applications. Proteins from leaves can contribute to a more complete use of resources and help to meet the increasing protein demand. Leaf protein extraction and purification is applied by other disciplines, such as proteomics.

  14. 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. (United States)

    Wada, Yasuaki; Lönnerdal, Bo


    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.

  15. Importance of extracellular proteins in maintaining structural integrity of aerobic granules. (United States)

    Xiong, Yanghui; Liu, Yu


    Aerobic granules developed through self-immobilization of microorganisms are compact and structured microbial consortia embedded in a matrix of extracellular polymeric substances (EPS). This study investigated the contribution of extracellular proteins (PN) to maintaining the structural integrity of aerobic granule. It was found that hydrolysis of PN induced by Proteinase K led to significant disintegration of aerobic granules, whereas a substantial reduction of extracellular polysaccharides (PS) was also observed. It was proposed that hydrolysis of extracellular proteins present in the EPS matrix of aerobic granules led to collapse of the EPS matrix, and subsequent disintegration of aerobic granule. These suggested that extracellular proteins would be essential for maintaining structural stability of EPS matrix of aerobic granules. In addition, it was revealed that production of signaling molecules, such as autoinducer-2 (AI-2) and N-acyl homoserine lactones (AHLs) was also inhibited probably due to hydrolysis of quorum sensing receptor proteins by Proteinase K. This in turn provided an additional explanation for the observed Proteinase K-triggered dispersal of aerobic granules.

  16. The effect of iodine salts on lipid oxidation and changes in nutritive value of protein in stored processed meats. (United States)

    Hęś, Marzanna; Waszkowiak, Katarzyna; Szymandera-Buszka, Krystyna


    The aim was to assess the effect of iodine salts (KI or KIO(3)) on lipid oxidation as well as changes in the availability of lysine and methionine and protein digestibility in frozen-stored processed meats. Three types of iodine salt carriers were used: table salt, wheat fiber and soy protein isolate. The results showed no catalytic effect of iodine salts on lipid oxidation in stored processed meats. The application of a protein isolate and wheat fiber resulted in the inhibition of lipid oxidation in meatballs. During storage of meat products the contents of available lysine and methionine as well as protein digestibility were decreased. The utilization of wheat fiber as an iodine salt carrier had a significant effect on the reduction of lysine losses. No protective properties were found for the wheat fiber or soy protein isolate towards methionine.

  17. Modulation of microRNA processing by mismatch repair protein MutLα

    Institute of Scientific and Technical Information of China (English)

    Guogen Mao; Sanghee Lee; Janice Ortega; Liya Gu; Guo-Min Li


    MicroRNAs (miRNAs) are critical post-transcriptional regulators and are derived from hairpnn-shaped primary transcripts via a series of processing steps.However,how the production of individual miRNAs is regulated remains largely unknown.Similarly,loss or overexpression of the key mismatch repair protein MutLα (MLH1-PMS2 heterodimer) leads to genome instability and tumorigenesis,but the mechanisms controlling MutLαt expression are unknown.Here we demonstrate in vitro and in vivo that MLH1 and miR-422a participate in a feedback loop that regulates the level of both molecules.Using a defined in-vitro miRNA processing system,we show that MutLαt stimulates the conversion of pri-miR-422a to pre-miR-422a,as well as the processing of other miRNAs tested,implicating MutLα as a general stimulating factor for miRNA biogenesis.This newly identified MutLαα function requires its ATPase and pri-miRNA binding activities.In contrast,miR-422a downregulates MutLα levels by suppressing MLH1 expression through base pairing with the MLH1 3'-untranslated region.A model depicting this feedback mechanism is discussed.

  18. Nucleic acid and protein elimination during the sugar manufacturing process of conventional and transgenic sugar beets. (United States)

    Klein, J; Altenbuchner, J; Mattes, R


    The fate of cellular DNA during the standard purification steps of the sugar manufacturing process from conventional and transgenic sugar beets was determined. Indigenous nucleases of sugar beet cells were found to be active during the first extraction step (raw juice production) which was carried out at 70 degrees C. This and the consecutive steps of the manufacturing process were validated in terms of DNA degradation by competitive PCR of added external DNA. Each step of the process proved to be very efficient in the removal of nucleic acids. Taken together, the purification steps have the potential to reduce the amount of DNA by a factor of > 10(14), exceeding by far the total amount of DNA present in sugar beets. Furthermore, the gene products of the transgenes neomycin phosphotransferase and BNYVV (rhizomania virus) coat protein CP21 were shown to be removed during the purification steps, so that they could not be detected in the resulting white sugar. Thus, sugar obtained from conventional and transgenic beets is indistinguishable or substantially equivalent with respect to purity.

  19. Single-Nucleotide Polymorphism of PPARγ, a Protein at the Crossroads of Physiological and Pathological Processes (United States)

    Petrosino, Maria; Lori, Laura; Pasquo, Alessandra; Lori, Clorinda; Consalvi, Valerio; Minicozzi, Velia; Morante, Silvia; Laghezza, Antonio; Giorgi, Alessandra; Capelli, Davide; Chiaraluce, Roberta


    Genome polymorphisms are responsible for phenotypic differences between humans and for individual susceptibility to genetic diseases and therapeutic responses. Non-synonymous single-nucleotide polymorphisms (nsSNPs) lead to protein variants with a change in the amino acid sequence that may affect the structure and/or function of the protein and may be utilized as efficient structural and functional markers of association to complex diseases. This study is focused on nsSNP variants of the ligand binding domain of PPARγ a nuclear receptor in the superfamily of ligand inducible transcription factors that play an important role in regulating lipid metabolism and in several processes ranging from cellular differentiation and development to carcinogenesis. Here we selected nine nsSNPs variants of the PPARγ ligand binding domain, V290M, R357A, R397C, F360L, P467L, Q286P, R288H, E324K, and E460K, expressed in cancer tissues and/or associated with partial lipodystrophy and insulin resistance. The effects of a single amino acid change on the thermodynamic stability of PPARγ, its spectral properties, and molecular dynamics have been investigated. The nsSNPs PPARγ variants show alteration of dynamics and tertiary contacts that impair the correct reciprocal positioning of helices 3 and 12, crucially important for PPARγ functioning. PMID:28208577

  20. Simultaneous production of trehalose, bioethanol, and high-protein product from rice by an enzymatic process. (United States)

    Chang, Shu-Wei; Chang, Wei-Hsin; Lee, Maw-Rong; Yang, Tzung-Jie; Yu, Nu-Yi; Chen, Chin-Shuh; Shaw, Jei-Fu


    Rice is a starch-rich raw material that can be used for trehalose production. It can be hydrolyzed with alpha-amylase, beta-amylase, and pullulanase to produce high-maltose content of rice saccharified solution for bioconversion of maltose into trehalose by trehalose synthase (TSase). For this purpose, an efficient enzymatic procedure has been successfully developed to simultaneously produce value-added trehalose, bioethanol, and high-protein product from rice as substrate. The highest maltose yield produced from the liquefied rice starch hydrolysate was 82.4 +/- 2.8% at 50 degrees C and pH 5.0 for 21-22 h. The trehalose conversion rate can reach at least 50% at 50 degrees C and pH 5.0 for 20-24 h by a novel thermostable recombinant Picrophilus torridus trehalose synthase (PTTS). All residual sugar, except trehalose, can be fully hydrolyzed by glucoamylase into glucose for further bioethanol production. The insoluble byproduct containing high yields of protein (75.99%) and dietary fiber (14.01%) can be processed as breakfast cereal product, health food, animal forage, etc. The conversion yield of bioethanol was about 98% after 64 h of fermentation time by Saccharomyces cerevisiae without any artificial culture solution addition. Ethanol can easily be separated from trehalose by distillation with a high recovery yield and purity of crystalline trehalose of 92.5 +/- 8.7% and 92.3%, respectively.

  1. 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))


    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.

  2. Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics. (United States)

    Fang, Jing; Nevin, Philip; Kairys, Visvaldas; Venclovas, Ceslovas; Engen, John R; Beuning, Penny J


    The relationship between protein sequence, structure, and dynamics has been elusive. Here, we report a comprehensive analysis using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics.

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

    Directory of Open Access Journals (Sweden)

    Chotipuntu, P.


    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.

  4. Different forms of MARCKS protein are involved in memory formation in the learning process of imprinting. (United States)

    Solomonia, Revaz O; Apkhazava, David; Nozadze, Maia; Jackson, Antony P; McCabe, Brian J; Horn, Gabriel


    There is strong evidence that a restricted part of the chick forebrain, the IMM (formerly IMHV), stores information acquired through the learning process of visual imprinting. Twenty-four hours after imprinting training, a learning-specific increase in amount of myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is known to occur in the homogenate fraction of IMM. We investigated the two components of this fraction, membrane-bound and cytoplasmic-phosphorylated MARCKS. In IMM, amount of membrane-bound MARCKS, but not of cytoplasmic-phosphorylated MARCKS, increased as chicks learned. No changes were observed for either form of MARCKS in PPN, a control forebrain region. The results indicate that there is a learning-specific increase in membrane-bound, non-phosphorylated MARCKS 24 h after training. This increase might contribute to stabilization of synaptic morphology.

  5. Efficient biological process characterization by definitive-screening designs: the formaldehyde treatment of a therapeutic protein as a case study. (United States)

    Erler, Axel; de Mas, Nuria; Ramsey, Philip; Henderson, Grant


    As part of the process-characterization campaign of a candidate vaccine product, a recently developed class of three-level designs-definitive-screening designs-was employed to select a quadratic model that describes the effect of six input process parameters, including protein concentration, formaldehyde-to-protein ratio, lysine concentration, reaction duration, pH, and reaction temperature, on a formylation protein-crosslinking reaction. This design requires only 17 experimental runs. The resulting model was then used to simulate 10,000 runs that account for the variability in the inputs expected on manufacturing scale. The extent of protein polymerization was predicted to be within specifications for all simulated runs, demonstrating the robustness of the unit operation for subsequent process validation and future commercial manufacturing.


    Directory of Open Access Journals (Sweden)

    A. A. Shevtsov


    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.

  7. Effect of two-step functionalization of Ti by chemical processes on protein adsorption (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Rotwein Peter


    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.

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

    Directory of Open Access Journals (Sweden)

    Gayol, M. F.


    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

  10. Influence of system and process parameters on partitioning of cheese whey proteins in aqueous two-phase systems. (United States)

    Rito-Palomares, M; Hernandez, M


    A practical study is described to characterise some problems encountered in the application of aqueous two-phase systems (ATPS) to protein recovery. These factors include practical design of extraction stages and the impact of ATPS compounding methods and biological suspension upon process performance. They were addressed using the recovery of whey proteins as a model. The known effects of system parameters (i.e. tie-line length, volume ratio and system pH) were exploited to define the specific operating conditions of a two-stage ATPS process for the recovery of whey proteins. The partition of whey proteins in ATPS assembled using different methods resulted in changes in the partition coefficient of the proteins. Such changes were associated with the initial location of the proteins in the polymer or salt-rich solutions of the ATPS. Cheese whey loaded into the ATPS caused the displacement of the binodal curve from the origin. Such behaviour was attributed to the residual fat present in the whey. These findings highlight those factors perceived as negative constraints on the wider adoption of ATPS processes for protein recovery from complex biological systems.

  11. Molecularly imprinted polymer grafted on polysaccharide microsphere surface by the sol-gel process for protein recognition. (United States)

    Li, Feng; Li, Jing; Zhang, Shusheng


    An interfacial organic-inorganic hybridization concept was applied to the preparation of a new spherical imprinted material for protein recognition. The functional biopolymer chitosan (CS), shaped as microsphere and high-density cross-linked, constituted of the polysaccharide core for surface imprinting. After the model template protein, bovine serum albumin, was covalently immobilized by forming imine bonds with the functional amine groups of CS, two kinds of organic siloxane (3-aminopropyltrimethoxysiloxane: APTMS, and tetraethoxysiloxane: TEOS) assembled and polymerized on the polysaccharide-protein surface via sol-gel process in aqueous solution at room temperature. After template removal, the protein-imprinted sol-gel surface exhibited a prevalent preference for the template protein in adsorption experiments, as compared with four contrastive proteins. Bioinformatics methods were also employed to investigate the imprinting process and the recognition effect. The influence of siloxane type, pH, siloxane/water ratio on template removal and recognition selectivity was assessed. Under optimized imprinting conditions, a large quantity of well-distributed pores was observed on the immobilized-template imprinted surface. The surface-imprinted adsorbent offered a fast kinetics for template re-adsorption and could be reused. Compared with the imprinted material prepared with free-template, material prepared with immobilized-template possessed higher adsorption capacity towards template protein. Easy preparation of the described imprinted material, high affinity and good reusability make this approach attractive and broadly applicable in biotechnology for down-stream processing and biosensor.

  12. Scale-up of the process to obtain functional ingredients based in plasma protein concentrates from porcine blood. (United States)

    Parés, Dolors; Toldrà, Mònica; Saguer, Elena; Carretero, Carmen


    The feasibility of a scaled-up process to obtain two protein concentrates from porcine blood plasma, i.e. serum and albumin, for use as functional food ingredients was assessed. The process consisted of fractionating plasma proteins by salting out, concentrating and purifying fractions by means of membrane technology, and subsequently dehydrating through spray-drying. The fractionation process allowed a good isolation of the desired proteins, which were then concentrated and desalted in a tangential flow filtration (TFF) process combining ultra and diafiltration. Purification, pre-concentration and dehydration were successfully achieved. The functional properties of dehydrated serum and albumin were determined. As compared to the same hemoderivatives obtained by a lab-scale production system, serum maintained the gelling properties; albumin exhibited similar foaming properties; and both serum and albumin concentrates showed slightly improved emulsifying properties.

  13. A process efficiency assessment of serum protein removal from milk using ceramic graded permeability microfiltration membrane. (United States)

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


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

  14. Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein. (United States)

    Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence


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

  15. RadA: A protein involved in DNA damage repair processes of Deinococcus radiodurans R1

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qing; ZHANG Xinjue; XU Hong; XU Bujin; HUA Yuejin


    RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein superfamily found in bacteria, archaea and eukarya. In Archaea, it plays a critical role in homologous recombination process due to its RecA-like function. In Escherichia coli, it takes part in conjugational recombination and DNA repair but is not as important as that of archaea. Using PSI-BLAST searches, we found that Deinococcus radiodurans RadA had a higher similarity to that of bacteria than archaea and eukarya. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet, but had no effect on the resistance to hydrogen peroxide. Complementation of the radA disruptant by both E. coli radA and D.radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype togamma and UV radiation than that of the radA mutant,while the absence of the Lon protease domain exhibited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.

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

    Directory of Open Access Journals (Sweden)

    G. Vonghia


    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...

  17. Gli2 and Gli3 Localize to Cilia and Require the Intraflagellar Transport Protein Polaris for Processing and Function

    Energy Technology Data Exchange (ETDEWEB)

    Michaud III, Edward J [ORNL; Haycraft, Courtney J [University of Alabama, Birmingham; Aydin Son, Yesim [University of Tennessee, Knoxville (UTK); Zhang, Qihong [University of Alabama, Birmingham; Yoder, Bradley [University of Alabama, Birmingham


    Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh)signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

  18. Gli2 and gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.

    Directory of Open Access Journals (Sweden)


    Full Text Available Intraflagellar transport (IFT proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

  19. 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;


    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...... 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...

  20. Composition of proteins in okara as a byproduct in hydrothermal processing of soy milk. (United States)

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


    Protein quality, based on its subunit composition, in okara obtained as a byproduct during hydrothermal cooking of soy milk was assessed. The composition of 7S and 11S protein fractions was correlated with the physicochemical properties of protein in okara produced from six soybean varieties. The basic 7S globulin (Bg7S) and 11S protein were two main proteins in okara. Investigated soybean genotypes produced okara with mainly acidic A(5) and basic B(1,2,4) polypeptides of 11S proteins. Soybean 11S content was not an indicator of okara protein recovery or extractability. Of all tested relationships, extractable soluble protein content of okara was influenced only by soybean Bg7S (r = 0.86; p soybeans (r = 0.81; p functional ingredient.

  1. Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis. (United States)

    Robinson, Mark W; Massie, Diane H; Connolly, Bernadette


    The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.

  2. Freeze-Drying Above the Glass Transition Temperature in Amorphous Protein Formulations While Maintaining Product Quality and Improving Process Efficiency. (United States)

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


    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.

  3. Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

    NARCIS (Netherlands)

    Freydell, E.J.; Wielen, van der L.A.M.; Eppink, M.H.M.; Ottens, M.


    Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be ref

  4. Influence of protein isolate from lupin seeds (Lupinus albus ssp. Graecus) on processing and quality characteristics of frankfurters. (United States)

    Alamanou, S; Bloukas, J G; Paneras, E D; Doxastakis, G


    Lupin protein isolate (92% protein) from seeds of Lupinus albus ssp. Graecus (LSPI) was used as powder ingredient for the manufacture of frankfurters at levels 0, 1, 2 and 3% of the formulation weight. Additional 1% water was added during batter formulation to each 1% protein used. LSPI increased (P LSPI level resulted in higher (P LSPI. Frankfurters with 3% LSPI were judged as unacceptable. Incorporation of LSPI at 1% level either in hydrated form or as stabilizer in pre-emulsified fat improved the processing characteristics and overall acceptability of frankfurters made with LSPI as powder ingredient and did not affect the color and texture.

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

    DEFF Research Database (Denmark)

    Liu, Guanchen

    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...... these interactions affect final acidified milk products. By detecting the properties of the whey protein aggregates, MWP and NWP showed low native whey protein content, low free thiol content and high surface hydrophobicity and were relatively stable at high temperature in the 5 % pure dispersions. When MWP and NWP...... with other proteins present and resulted in a protein network with low connectivity in the resulting gels. Increasing the casein/whey protein ratio did not decrease the gel strength in the acidified milk model systems with added whey protein aggregates. The results of this study highlighted the influences...

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

    Directory of Open Access Journals (Sweden)

    Dell'Orto V.


    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. Subcellular localization of cytoplasmic lattice-associated proteins is dependent upon fixation and processing procedures. (United States)

    Morency, Eric; Anguish, Lynne; Coonrod, Scott


    We and others have recently demonstrated by immuno-EM and mutation analysis that two oocyte-restricted maternal effect genes, PADI6 and MATER, localize, in part, to the oocyte cytoplasmic lattices (CPLs). During these ongoing studies, however, we found that the localization of these factors by confocal immunofluorescence (IF) analysis can vary dramatically depending upon how the oocytes and embryos are processed, with the localization pattern sometimes appearing more uniformly cytoplasmic while at other times appearing to be primarily cortical. We set out to better understand this differential staining pattern by testing a range of IF protocol parameters, changing mainly time and temperature conditions of the primary antibody solution incubation, as well as fixation methods. We found by confocal IF whole mount analysis that PADI6 and MATER localization in germinal vesicle stage oocytes is mainly cytoplasmic when the oocytes are fixed and then incubated with primary antibodies at room temperature for 1 hour, while the localization of these factors is largely limited to the cortex when the oocytes are fixed and incubated in primary antibody at 4 °C overnight. We then probed sections of fixed/embedded ovaries and isolated two-cell embryos with specific antibodies and found that, under these conditions, PADI6 and MATER were again primarily cytoplasmically localized, although the staining for these factors is slightly more cortical at the two-cell stage. Taken together, our results suggest that the localization of CPL-associated proteins by confocal IF is particularly affected by processing conditions. Further, based on our current observations, it appears that PADI6 and MATER are primarily distributed throughout the cytoplasm as opposed to the oocyte subcortex.

  8. Novel effects of FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone] on amyloid precursor protein processing. (United States)

    Connop, B P; Thies, R L; Beyreuther, K; Ida, N; Reiner, P B


    Amyloidogenic processing of the beta-amyloid precursor protein (APP) has been implicated in the pathology of Alzheimer's disease. Because it has been suggested that catabolic processing of the APP holoprotein occurs in acidic intracellular compartments, we studied the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and the H+-ATPase inhibitor bafilomycin A1 on APP catabolism in human embryonic kidney 293 cells expressing either wild-type or "Swedish" mutant APP. Unlike bafilomycin A1, which inhibits beta-amyloid production in cells expressing mutant but not wild-type APP, FCCP inhibited beta-amyloid production in both cell types. Moreover, the effects of FCCP were independent of alterations in total cellular APP levels or APP maturation, and the concentrations used did not alter either cellular ATP levels or cell viability. Bafilomycin A1, which had no effect on beta-amyloid production in wild-type cells, inhibited endocytosis of fluorescent transferrin, whereas concentrations of FCCP that inhibited beta-amyloid production in these cells had no effect on endosomal function. Thus, in wild-type-expressing cells it appears that the beta-amyloid peptide is not produced in the classically defined endosome. Although bafilomycin A1 decreased beta-amyloid release from cells expressing mutant APP but not wild-type APP, it altered lysosomal function in both cell types, suggesting that in normal cells beta-amyloid is not produced in the lysosome. Although inhibition of beta-amyloid production by bafilomycin A1 in mutant cells may occur via changes in endosomal/lysosomal pH, our data suggest that FCCP inhibits wild-type beta-amyloid production by acting on a bafilomycin A1-insensitive acidic compartment that is distinct from either the endosome or the lysosome.

  9. Fluorescence monitoring of the effect of oxidized lipids on the process of protein fibrillization (United States)

    Vus, Kateryna; Sood, Rohit; Gorbenko, Galyna; Kinnunen, Paavo


    The kinetics of lysozyme and insulin amyloid formation in the presence of the oxidized phospholipids (oxPLs) was investigated using Thioflavin T fluorescence assay. The kinetic parameters of fibrillization process (lag time and apparent rate constant) have been determined upon varying the following experimental parameters: the type of lipid assemblies (premicellar aggregates and lipid bilayer vesicles), pH, temperature and lipid-to-protein molar ratio. It was found that oxPLs premicellar aggregates induced the more pronounced increase of the maximum Thioflavin T fluorescence, which is proportional to the extent of fibril formation, compared to the vesicles composed of the oxidized and unoxidized lipids. In contrast, the oxPLs, used as dispersions or included into vesicles, inhibited fibril nucleation and elongation under near-physiological conditions in vitro compared to liposomes containing unoxidized lipids. The results obtained provide deeper insight into the molecular mechanisms of the oxidative stress-modulated conformational diseases, and could be employed for the anti-amyloid drug development.

  10. High hydrostatic pressure processing: a method having high success potential in pollen protein extraction (United States)

    Murat Altuner, Ergin; Çeter, Talip; Alpas, Hami


    Even a single peptide that is present in the pollen wall and cytoplasm could cause pollen allergy. To produce skin-prick test kits, the first step is the extraction of these molecules. In this study, Cedrus atlantica pollens were subjected to 220 and 330 MPa for 10 and 30 min in order to extract these molecules. After high hydrostatic pressure processing (HHPP), the total amounts of proteins (TAPs) are measured and compared with the results of the conventional extraction method (CEM). As a result, the TAPs extracted by HHPP is 18.0210 μ g/mL at 220 MPa for 10 min, 22.5770 μ g/mL at 220 MPa for 30 min, 23.3810 μ g/mL at 330 MPa for 10 min and 25.9270 μ g/mL at 330 MPa for 30 min, while this is 1.9460 μ g/mL in 24 h by the CEM. In addition to these results, visual pollen deformation and eruption, pollen wall and surface damage have also been observed.

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

    Directory of Open Access Journals (Sweden)

    Fumière O.


    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.

  12. Production of coconut protein powder from coconut wet processing waste and its characterization. (United States)

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


    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.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  14. A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic Swine Origin Influenza a virus Hemagglutinin protein. (United States)

    Athmaram, T N; Singh, Anil Kumar; Saraswat, Shweta; Srivastava, Saurabh; Misra, Princi; Kameswara Rao, M; Gopalan, N; Rao, P V L


    The present Influenza vaccine manufacturing process has posed a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza. New vaccine strategies are therefore needed that can accelerate the vaccine production. Pichia offers several advantages for rapid and economical bulk production of recombinant proteins and, hence, can be attractive alternative for producing an effective influenza HA based subunit vaccine. The recombinant Pichia harboring the transgene was subjected to fed-batch fermentation at 10 L scale. A simple fermentation and downstream processing strategy is developed for high-yield secretory expression of the recombinant Hemagglutinin protein of pandemic Swine Origin Influenza A virus using Pichia pastoris via fed-batch fermentation. Expression and purification were optimized and the expressed recombinant Hemagglutinin protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot and MALDI-TOF analysis. In this paper, we describe a fed-batch fermentation protocol for the secreted production of Swine Influenza A Hemagglutinin protein in the P. pastoris GS115 strain. We have shown that there is a clear relationship between product yield and specific growth rate. The fed-batch fermentation and downstream processing methods optimized in the present study have immense practical application for high-level production of the recombinant H1N1 HA protein in a cost effective way using P. pastoris.

  15. 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


    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.

  16. 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


    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.


    Directory of Open Access Journals (Sweden)

    Bernd Stanislawski


    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.

  18. RP-HPLC and chemometrics for wheat flour protein characterisation in an industrial bread-making process monitoring context. (United States)

    Li Vigni, Mario; Baschieri, Carlo; Marchetti, Andrea; Cocchi, Marina


    In the baking industry, a difficult task is to keep the quality perceived by the consumer as constant as possible, given the inner variability of flour, e.g. due to different wheat mixtures, harvesting time, etc. Here, we evaluated the influence of flour batches properties on bread quality, considering an industrial bread making process. In particular, flour composition in terms of protein fractions (gliadins, glutenins) has been determined by means of RP-HPLC, to assess the inter- and intra-batch variability of flour mixtures deliveries at a baking plant. Multivariate data analysis allowed evaluation of correlation between flour protein composition and technological properties. A great variability within different deliveries of a same flour batch emerged, as well as a considerable seasonal variability. Correlation models among protein sub-fractions, technological properties and bread quality are difficult to establish; however, the role of the protein profile on flour behaviour in bread making could be highlighted.

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


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


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

  20. The minute virus of canines (MVC) NP1 protein governs the expression of a subset of essential NS proteins via its role in RNA processing. (United States)

    Fasina, Olufemi O; Stupps, Stephanie; Figueroa-Cuilan, Wanda; Pintel, David J


    Parvoviruses use a variety of means to control the expression of their compact genomes. The Bocaparvovirus Minute Virus of Canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional non-structural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply-spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino terminus of these proteins into the NP1 open reading frame, and splice-site mutations that prevented their expression inhibited virus replication in a host-cell dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing.IMPORTANCE The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the Bocaparvovirus genus. It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show here that NP1 also controls expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins are together required for virus replication in a host-cell dependent manner.

  1. Identification and Quantitative Analysis of Significantly Accumulated Proteins During the Arabidopsis Seedling De-etiolation Process

    Institute of Scientific and Technical Information of China (English)

    Bai-Chen Wang; Ying-Hong Pan; Da-Zhe Meng; Yu-Xian Zhu


    Proteomic analysis was performed on seedlings after different light treatments. A total of (1 350±31) protein spots was separated and visualized on each silver nitrate-stained two-dimensional gel using protein samples prepared from light-grown or etiolated seedlings with or without 6-9 h light treatment. Twenty-five protein spots (encoded by 19 genes) that were significantly accumulated upon light treatment were identified using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method. Functional proteomics indicated that these proteins involved mainly in chloroplast development, energy metabolism,cell cycle progression and membrane electron transport. For 18 of the protein-coding genes we identified through an internet search, the transcript levels of 17 genes matched roughly with their protein content in etiolated and green seedlings, suggesting that these genes were regulated by light mainly at the transcriptional level. Despite a very significant increase in the amount of proteins upon light treatment, similar RNA levels were found in dark-grown or green seedlings for the carbonic anhydrase gene At3g05100, indicating a possible post-transcriptional regulatory mechanism. Elucidation of light-induced protein accumulation will undoubtedly enhance our understanding of plant photomorphogenesis.

  2. Characterization and value-added utilization of proteins extracted from the by-products from catfish fillet processing plant (United States)

    Channel catfish farming is the most important warm water aquaculture in the Southeastern United States. The by-products, including heads, skin, bone frame and viscera, account for 55-65% of the whole fish mass after fillet processing. The by-products contain 35% of protein on a dry basis, and may be...

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

    DEFF Research Database (Denmark)

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


    and levels of retromer proteins are altered in AD. Here we report that sorLA and retromer functionally interact in neurons to control trafficking and amyloidogenic processing of APP. We have identified a sequence (FANSHY) in the cytoplasmic domain of sorLA that is recognized by the VPS26 subunit...

  4. Effect of processing methods on the mechanical properties of natural rubber filled with stearic acid modified soy protein particles (United States)

    Natural rubber was reinforced with stearic acid modified soy protein particles prepared with a microfluidizing and ball milling process. Longer ball milling time tends to increase tensile strength of the rubber composites. Elastic modulus of the composites increased with the increasing filler concen...

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

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


    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.

  7. Genome-wide association study of CSF levels of 59 alzheimer's disease candidate proteins: significant associations with proteins involved in amyloid processing and inflammation.

    Directory of Open Access Journals (Sweden)

    John S K Kauwe


    Full Text Available Cerebrospinal fluid (CSF 42 amino acid species of amyloid beta (Aβ42 and tau levels are strongly correlated with the presence of Alzheimer's disease (AD neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC and Alzheimer's Disease Neuroimaging Initiative (ADNI, were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE, Chemokine (C-C motif ligand 2 (CCL2, Chemokine (C-C motif ligand 4 (CCL4, Interleukin 6 receptor (IL6R and Matrix metalloproteinase-3 (MMP3 with study-wide significant p-values (p<1.46×10-10 and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal

  8. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes. (United States)

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


    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.

  9. Azotobacter vinelandii metal storage protein: "classical" inorganic chemistry involved in Mo/W uptake and release processes. (United States)

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


    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

  10. 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: [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia)


    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.

  11. Gene p53 mutations, protein p53, and anti-p53 antibodies as biomarkers of cancer process. (United States)

    Lutz, Waldemar; Nowakowska-Swirta, Ewa


    The finding that gene mutations and changes in their expression form the basis of cancer processes, has prompted molecular epidemiologists to use biomarkers for detecting damaged genes or proteins synthesized under their control in easily available cellular material or systemic liquids. Mutations in the suppressor gen p53 are thought to be essential for cancer development. This gen is one of the most important regulators of transcription, cellular cycle, DNA repair and apoptosis detected till now. Inactivation of gene p53 leads to uncontrolled cell divisions, and further to transformation of normal cells into the carcinous ones. Observations that mutations in gene p53 appear under conditions of occupational and environmental exposures to chemical and physical carcinogens, such as vinyl chloride, radon, or aflatoxin B1, have proved to be of enormous importance for the occupational and environmental health. Changes in expression of gene p53, and also its mutations, cause variations of cellular protein p53 concentration. Higher cellular protein p53 levels are associated with increased protein transfer to the extracellular liquid and to blood. It has been observed that increased blood serum protein p53 concentrations may have a prognostic value in early diagnosis of lung cancer. The results of a number of studies confirm that accumulation of a mutated form of protein p53, and presumably also large quantities of wild forms of that protein in the cells, may be a factor that triggers the production of anti-p53 antibodies. Statistical analysis showed that anti-p53 antibodies can be regarded as a specific biomarker of cancer process. The prevalence of anti-p53 antibodies correlated with the degree of cancer malignancy. The increased incidence of anti-p53 antibodies was also associated with higher frequency of mutations in gene p53. There are some reports confirming that anti-p53 antibodies emerging in blood serum in the subclinical phase of cancer development may be

  12. Age-related changes of protein- and RNA-synthetic processes in experimental hyper- and hypothyroidism. (United States)

    Gromakova, I A; Zilberman, S T; Konovalenko, O A


    The rate of liver and plasma protein synthesis and the activity of liver RNA polymerases 1 and 2 were investigated in rats of various age under experimental hyper- and hypothyroidism. The rate of plasma protein synthesis decreased with age more dramatically than that of liver proteins. Hyper- and hypothyroidism exerted opposite effects on protein synthesis in rats: stimulation and inhibition, respectively. The manifestation of these effects was age related. The thyroid status of animals also influenced the balance of protein synthesis. Thyroxin administration caused preferential incorporation of a label into blood plasma proteins. Changes of thyroid status of old animals insignificantly affected the absolute values of the label incorporation into proteins and the ratio of the label incorporation into local and secreted liver proteins. Age-related decrease of total hepatic nuclear RNA-polymerase activity was due to reduction of the template-bound functionally active forms of RNA-polymerases 1 and 2. Administration of thyroxin caused initial redistribution of the enzyme activity between template-bound and free fractions accompanied by the increase of template bound RNA-polymerases. Prolonged hormonal stimulus also caused an increase of free RNA-polymerases, which reflects the increased synthesis of these enzymes. Mecrazolyl administration reduced the activity of RNA-polymerase 1 and 2. All age groups were characterized by preferential reduction of the bound form. RNA-polymerase 2 activity decreased to a greater extent than that of RNA-polymerase 1. The data suggest age-determined reactions of the body to altered thyroid status.

  13. 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.


    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.

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

    Directory of Open Access Journals (Sweden)

    Richard Park

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

  15. Determination of processed animal proteins, including meat and bone meal, in animal feed. (United States)

    Gizzi, Giséile; von Holst, Christoph; Baeten, Vincent; Berben, Gilbert; van Raamsdonk, Leo


    An intercomparison study was conducted to determine the presence of processed animal proteins (PAPs), including meat and bone meal (MBM) from various species, in animal feed. The performances of different methods, such as microscopy, polymerase chain reaction (PCR), immunoassays, and a protocol based on iquid chromatography (LC), were compared. Laboratories were asked to analyze for PAPs from all terrestrial animals and fish (total PAPs); mammalian PAPs; ruminant PAPs; and porcine PAPs. They were free to use their method of choice. In addition, laboratories using microscopy were asked to determine the presence of PAPs from terrestrial animals, which is applicable only to microscopy. For total PAPs microscopy, LC and some immunoassays showed sufficient results at a concentration as low as 0.1% MBM in the feed. In contrast, PCR was not fit for purpose. In differentiating between MBM from terrestrial animals and fishmeal, microscopy detected 0.5% of terrestrial MBM in feed in the presence of 5% fishmeal, but was less successful when the concentration of MBM from terrestrial animals was 0.1%. The animal-specific determination of MBM from mammals or, more specifically from either ruminants or pigs, by PCR showed poor results, as indicated by a high number of false-positive and false-negative results. The only PCR method that scored quite well was applied by a member of the organizer team of the study. Immunoassays scored much better than PCR, showing sufficient sensitivity but some deficiency in terms of specificity. The results also demonstrated that the reliable determination of MBM from ruminants has not been resolved, especially for low concentrations of MBM (0.1%) in feed. Comparison of the results for mammalian MBM from all methods indicated that, for control purposes, the immunoassay method, especially when applied as dipsticks, could be used as a rapid screening method combined with microscopy to confirm the positive samples. However, implementation of such a

  16. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein. (United States)

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D


    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc.

  17. Pumpkin (Cucurbita maxima) seed proteins: sequential extraction processing and fraction characterization. (United States)

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


    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.

  18. Formation of long-lived radicals on proteins by radical transfer from heme enzymes--a common process?

    DEFF Research Database (Denmark)

    Ostdal, H; Andersen, H J; Davies, Michael Jonathan


    albumin, indicating that the tertiary structure of the target protein plays an important role in determining the rate of radical transfer and/or the stability of the resultant species. These results are consistent with a mechanism for the HRP/H2O2/no free tyrosine system involving radical transfer...... to the albumin via the heme edge of the peroxidase. In contrast, albumin radical formation by the HRP/H2O2/free tyrosine system was only marginally affected by proteolysis, consistent with free tyrosine phenoxyl radicals being the mediators of radical transfer, without significant protein-protein interaction......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...

  19. 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;


    replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear...... the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms...... for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes....

  20. Investigate the Effect of Thawing Process on the Self-Assembly of Silk Protein for Tissue Applications (United States)

    Tran, Hien Anh; Huynh, Khon Chan; Vo, Toi Van


    Biological self-assembly is a process in which building blocks autonomously organize to form stable supermolecules of higher order and complexity through domination of weak, noncovalent interactions. For silk protein, the effect of high incubating temperature on the induction of secondary structure and self-assembly was well investigated. However, the effect of freezing and thawing on silk solution has not been studied. The present work aimed to investigate a new all-aqueous process to form 3D porous silk fibroin matrices using a freezing-assisted self-assembly method. This study proposes an experimental investigation and optimization of environmental parameters for the self-assembly process such as freezing temperature, thawing process, and concentration of silk solution. The optical images demonstrated the possibility and potential of −80ST48 treatment to initialize the self-assembly of silk fibroin as well as controllably fabricate a porous scaffold. Moreover, the micrograph images illustrate the assembly of silk protein chain in 7 days under the treatment of −80ST48 process. The surface morphology characterization proved that this method could control the pore size of porous scaffolds by control of the concentration of silk solution. The animal test showed the support of silk scaffold for cell adhesion and proliferation, as well as the cell migration process in the 3D implantable scaffold.

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

    DEFF Research Database (Denmark)

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


    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....

  2. Correlation between sequence, structure and function for trisporoid processing proteins in the model zygomycete Mucor mucedo. (United States)

    Ellenberger, Sabrina; Schuster, Stefan; Wöstemeyer, Johannes


    Terpenoids, steroids, carotenoids, phytoenes and other chemically related substance groups fulfill multiple functions in all realms of the organismic world. This analysis focuses on trisporoids that operate as pheromones in the phylogenetically ancient fungal group of mucoralean zygomycetes. Trisporoids serve as pheromones for recognizing complementary mating partners and for inducing the differentiation program towards sexual spore formation. Trisporoids are synthesized by oxidative degradation of β-carotene. Structurally, they are related to retinoids in mammals and abscisic acid in vascular plants. In order to evaluate evolutionary relationships between proteins involved in trisporoid binding and also for checking possibilities to recognize functionally related proteins by sequence and structure comparisons, we compared representative proteins of different origins. Towards this goal, we calculated three-dimensional structures for 4-dihydromethyltrisporate dehydrogenase (TSP1) and 4-dihydrotrisporin dehydrogenase (TSP2), the two proteins involved in trisporic acid synthesis that have unequivocally been correlated with their catalytic function for the model zygomycete Mucor mucedo. TSP1 is an aldo-keto reductase with a TIM-barrel structure, TSP2 belongs to short-chain dehydrogenases, characterized by a Rossmann fold. Evidently, functional conservation, even implying very similar substrates and identical cosubstrates of enzymes in a single organism, turns out to be essentially independent of basic protein structure. The binding sites for NADP and trisporoid ligands in the proteins were determined by docking studies, revealing those regions affecting substrate specificity. Despite the pronounced differences in amino acid sequence and tertiary structure, the surfaces around the active sites are comparable between TSP1 and TSP2. Two binding regions were identified, one sterically open and a second closed one. In contrast to TSP1, all docking models for TSP2 place the

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

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


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

  4. Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity

    NARCIS (Netherlands)

    Long, G.; Pan, X.; Vlak, J.M.


    The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common fe

  5. Experimental studies related to the origin of the genetic code and the process of protein synthesis - A review (United States)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.


    A survey is presented of the literature on the experimental evidence for the genetic code assignments and the chemical reactions involved in the process of protein synthesis. In view of the enormous number of theoretical models that have been advanced to explain the origin of the genetic code, attention is confined to experimental studies. Since genetic coding has significance only within the context of protein synthesis, it is believed that the problem of the origin of the code must be dealt with in terms of the origin of the process of protein synthesis. It is contended that the answers must lie in the nature of the molecules, amino acids and nucleotides, the affinities they might have for one another, and the effect that those affinities must have on the chemical reactions that are related to primitive protein synthesis. The survey establishes that for the bulk of amino acids, there is a direct and significant correlation between the hydrophobicity rank of the amino acids and the hydrophobicity rank of their anticodonic dinucleotides.

  6. Dysregulated striatal neuronal processing and impaired motor behavior in mice lacking huntingtin interacting protein 14 (HIP14.

    Directory of Open Access Journals (Sweden)

    Ana María Estrada-Sánchez

    Full Text Available Palmitoyl acyl transferases (PATs play a critical role in protein trafficking and function. Huntingtin interacting protein 14 (HIP14 is a PAT that acts on proteins associated with neuronal transmission, suggesting that deficient protein palmitoylation by HIP14, which occurs in the YAC128 model of Huntington's disease (HD, might have deleterious effects on neurobehavioral processing. HIP14 knockout mice show biochemical and neuropathological changes in the striatum, a forebrain region affected by HD that guides behavioral choice and motor flexibility. Thus, we evaluated the performance of these mice in two tests of motor ability: nest-building and plus maze turning behavior. Relative to wild-type controls, HIP14 knockout mice show impaired nest building and decreased turning in the plus maze. When we recorded the activity of striatal neurons during plus-maze performance, we found faster firing rates and dysregulated spike bursting in HIP14 knockouts compared to wild-type. There was also less correlated firing between simultaneously recorded neuronal pairs in the HIP14 knockouts. Overall, our results indicate that HIP14 is critically involved in behavioral modulation of striatal processing. In the absence of HIP14, striatal neurons become dysfunctional, leading to impaired motor behavior.

  7. Comparative Proteomic Analysis of Proteins Involved in the Tumorigenic Process of Seminal Vesicle Carcinoma in Transgenic Mice

    Directory of Open Access Journals (Sweden)

    Wei-Chao Chang


    Full Text Available We studied the seminal vesicle secretion (SVS of transgenic mice by using one-dimensional gel electrophoresis combined with LTQ-FT ICR MS analysis to explore protein expression profiles. Using unique peptide numbers as a cut-off criterion, 79 proteins were identified with high confidence in the SVS proteome. Label-free quantitative analysis was performed by using the IDEAL_Q software program. Furthermore, western blot assays were performed to validate the expression of seminal vesicle proteins. Sulfhydryl oxidase 1, glia-derived nexin, SVS1, SVS3, and SVS6 showed overexpression in SVS during cancer development. With high sequence similarity to human semenogelin, SVS2 is the most abundance protein in SVS and is dramatically decreased during the tumorigenic process. Our results indicate that these protein candidates could serve as potential targets for monitoring seminal vesicle carcinoma. Moreover, this information can provide clues for investigating seminal vesicle secretion-containing seminal plasma for related human diseases.

  8. Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach. (United States)

    Shukla, A A; Sorge, L; Boldman, J; Waugh, S


    The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies.

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

    NARCIS (Netherlands)

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


    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, a

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

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I


    resolution. (3) These proteins have no other cofactors except for the copper ions, thus the role of the polypeptide matrix can be addressed in a more straightforward manner. In azurins, the ET from the cystine (3-26) radical-ion produced by pulse-radiolytic reduction of this single disulfide bridge...

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

    NARCIS (Netherlands)

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


    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

  12. Value Added Processing of Aflatoxin Contaminated Peanut Meal: Aflatoxin Sequestration During Protein Extraction (United States)

    The efficacy of a bentonite clay, Astra-Ben 20A (AB20A), to sequester aflatoxin from contaminated (~110 ppb) peanut meal during protein extraction was studied. Aqueous peanut meal dispersions (10% w/w) were prepared varying pH, temperature, enzymatic hydrolysis conditions, and concentrations of AB2...

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

    DEFF Research Database (Denmark)

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

    model organisms’ shows that collecting unique annotation information from multiple sources significantly increases the protein annotation coverage in human, mouse, yeast, C. elegans and E. coli. A quantitative stable isotope labeling proteomics study comparing wild type C. elegans and a nuclear hormone...

  14. Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates

    NARCIS (Netherlands)

    Baclayon, Marian; van Ulsen, Peter; Mouhib, Halima; Shabestari, Maryam Hashemi; Verzijden, Timo; Abeln, Sanne; Roos, Wouter H; Wuite, Gijs J L


    The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively-stable in

  15. High productivity chromatography refolding process for Hepatitis B Virus X (HBx) protein guided by statistical design of experiment studies. (United States)

    Basu, Anindya; Leong, Susanna Su Jan


    The Hepatitis B Virus X (HBx) protein is a potential therapeutic target for the treatment of hepatocellular carcinoma. However, consistent expression of the protein as insoluble inclusion bodies in bacteria host systems has largely hindered HBx manufacturing via economical biosynthesis routes, thereby impeding the development of anti-HBx therapeutic strategies. To eliminate this roadblock, this work reports the development of the first 'chromatography refolding'-based bioprocess for HBx using immobilised metal affinity chromatography (IMAC). This process enabled production of HBx at quantities and purity that facilitate their direct use in structural and molecular characterization studies. In line with the principles of quality by design (QbD), we used a statistical design of experiments (DoE) methodology to design the optimum process which delivered bioactive HBx at a productivity of 0.21 mg/ml/h at a refolding yield of 54% (at 10 mg/ml refolding concentration), which was 4.4-fold higher than that achieved in dilution refolding. The systematic DoE methodology adopted for this study enabled us to obtain important insights into the effect of different bioprocess parameters like the effect of buffer exchange gradients on HBx productivity and quality. Such a bioprocess design approach can play a pivotal role in developing intensified processes for other novel proteins, and hence helping to resolve validation and speed-to-market challenges faced by the biopharmaceutical industry today.

  16. New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation. (United States)

    González-Martínez, Alejandro; Calderón, Kadiya; González-López, Jesús


    High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.

  17. Application of Quality by Design to the characterization of the cell culture process of an Fc-Fusion protein. (United States)

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


    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.

  18. Information processing and intracellular 'neural' (protein) networks: considerations regarding the diffusion-based hypothesis of Bray. (United States)

    Agutter, P S; Wheatley, D N


    The possibility that the disposition of subsets of proteins within the cell can retain memory traces and may act therefore in a computationial role has been advanced and more recently refined by Bray (Nature (1995) 376, 307-312). The proposition is not without its merits but inevitably has a number of associated difficulties, some of which are discussed in this article. These relate to the nature of the computational units envisaged (analog vs digital), their limitations in the number of stable patterns they can accommodate, the reliance on diffusion at the molecular level as the 'governing principle', and the complication of the turnover of proteins through degradative mechanisms. These issues suggest that certain modifications of the original model are required.

  19. Preparation and characterization of whey protein hydrolysates: applications in industrial whey bioconversion processes. (United States)

    Perea, A; Ugalde, U; Rodriguez, I; Serra, J L


    A whey protein hydrolysate was prepared by incubation of reconstituted whey or a whey protein concentrate with Alcalase 0.6L. The proteolytic degradation of alpha-lactalbumin and beta-lactoglobulin initially resulted in 6-kDa and, later, 2.5-kDa degradation products, quickly followed by the appearance of multiple peptides of 1 kDa or smaller. The hydrolysate showed a steady increase in solubility and a biphasic change in foaming characteristics with decreasing peptide size. At the highest degree of hydrolysis achieved (22%), the majority of the peptides were smaller than 1 kDa and could be efficiently assimilated by the yeast Kluyveromyces marxianus growing in a defined medium.

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

    LENUS (Irish Health Repository)

    Bazzi, M D


    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.

  1. Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. (United States)

    Jahic, Mehmedalija; Gustavsson, Malin; Jansen, Ann-Katrin; Martinelle, Mats; Enfors, Sven-Olof


    A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g x l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degrees C during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g x l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

  2. 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.

  3. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid. (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi


    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  4. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis (United States)

    Yang, Leiyun; Yang, Fen; Wang, Yi; Zhu, Jian-Kang; Hua, Jian


    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. PMID:27138552

  5. Proteomic analysis of processing by-products from canned and fresh tuna: identification of potentially functional food proteins. (United States)

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


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

  6. Protein encapsulated core-shell structured particles prepared by coaxial electrospraying: investigation on material and processing variables. (United States)

    Zamani, Maedeh; Prabhakaran, Molamma P; Thian, Eng San; Ramakrishna, Seeram


    Biodegradable polymeric particles have been extensively investigated for controlled drug delivery of various therapeutic agents. 'Coaxial' electrospraying was successfully employed in this study, to fabricate core-shell PLGA particles containing bovine serum albumin (BSA) as the model protein, and the results were also compared to particles prepared by 'emulsion' electrospraying. Two different molecular weights of PLGA were employed to encapsulate the protein. Solution properties and processing parameters were found to influence the morphology of the core-shell particles. Depending on the type of solvent used to dissolve the polymer as well as the polymer concentration and molecular weight, the mean diameter of the particles varied between 3.0 to 5.5 μm. Fluorescence microscopic analysis of the electrosprayed particles using FITC-conjugated BSA demonstrated the core-shell structure of the developed particles. The encapsulation efficiency and release behavior of BSA was influenced by shell:core feeding ratio, protein concentration, and the electrospraying method. The encapsulation efficiency of BSA within the core-shell particles of high and low molecular weight PLGA was found 15.7% and 25.1% higher than the emulsion electrosprayed particles, respectively. Moreover, the total amount of BSA released from low molecular weight PLGA particles was significantly higher than high molecular weight PLGA particles within 43 days of release studies, with negligible effect on encapsulation efficiency. The technique of coaxial electrospraying has high potential for encapsulation of susceptible protein-based therapeutic agents such as growth factors for multiple drug delivery applications.

  7. Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin. (United States)

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


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

  8. Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods. (United States)

    Santi, Lucélia; Silva, Walter O B; Pinto, Antônio F M; Schrank, Augusto; Vainstein, Marilene H


    Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.

  9. Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula. (United States)

    Lyon, David; Castillejo, Maria Angeles; Mehmeti-Tershani, Vlora; Staudinger, Christiana; Kleemaier, Christoph; Wienkoop, Stefanie


    Climate change in conjunction with population growth necessitates a systems biology approach to characterize plant drought acclimation as well as a more thorough understanding of the molecular mechanisms of stress recovery. Plants are exposed to a continuously changing environment. Extremes such as several weeks of drought are followed by rain. This requires a molecular plasticity of the plant enabling drought acclimation and the necessity of deacclimation processes for recovery and continuous growth.During drought stress and subsequent recovery, the metabolome and proteome are regulated through a sequence of molecular processes including synthesis and degradation and molecular interaction networks are part of this regulatory process. In order to study this complex regulatory network, a comprehensive analysis is presented for the first time, investigating protein turnover and regulatory classes of proteins and metabolites during a stress recovery scenario in the model legume Medicago truncatula The data give novel insights into the molecular capacity and differential processes required for acclimation and deacclimation of severe drought stressed plants.Functional cluster and network analyses unraveled independent regulatory mechanisms for stress and recovery with different dynamic phases that during the course of recovery define the plants deacclimation from stress. The combination of relative abundance levels and turnover analysis revealed an early transition phase that seems key for recovery initiation through water resupply and is independent from renutrition. Thus, a first indication for a metabolite and protein-based load capacity was observed necessary for the recovery from drought, an important but thus far ignored possible feature toward tolerance. The data indicate that apart from the plants molecular stress response mechanisms, plasticity may be related to the nutritional status of the plant prior to stress initiation. A new perspective and possible new

  10. 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)


    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

  11. New learning while consolidating memory during sleep is actively blocked by a protein synthesis dependent process (United States)

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


    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: PMID:27919318

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

    Directory of Open Access Journals (Sweden)

    Beat eSchwaller


    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.

  13. Elg1, the major subunit of an alternative RFC complex, interacts with SUMO-processing proteins. (United States)

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


    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.

  14. New approach for the quantification of processed animal proteins in feed using light microscopy. (United States)

    Veys, P; Baeten, V


    A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed.

  15. The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane. (United States)

    Russo, Alessandra; Bonci, Paola; Bonci, Paolo


    The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.

  16. A defect in the RNA-processing protein HNRPDL causes limb-girdle muscular dystrophy 1G (LGMD1G). (United States)

    Vieira, Natássia M; Naslavsky, Michel S; Licinio, Luciana; Kok, Fernando; Schlesinger, David; Vainzof, Mariz; Sanchez, Nury; Kitajima, João Paulo; Gal, Lihi; Cavaçana, Natale; Serafini, Peter R; Chuartzman, Silvia; Vasquez, Cristina; Mimbacas, Adriana; Nigro, Vincenzo; Pavanello, Rita C; Schuldiner, Maya; Kunkel, Louis M; Zatz, Mayana


    Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in S. cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA-processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for muscular dystrophies.

  17. Evidence supporting the role of calpain in the α-processing of amyloid-β precursor protein. (United States)

    Nguyen, Huey T; Sawmiller, Darrell R; Wu, Qi; Maleski, Jerome J; Chen, Ming


    Amyloid plaques are a hallmark of the aging and senile dementia brains, yet their mechanism of origins has remained elusive. A central issue is the regulatory mechanism and identity of α-secretase, a protease responsible for α-processing of amyloid-β precursor protein (APP). A remarkable feature of this enzyme is its high sensitivity to a wide range of cellular stimulators, many of which are agonists for Ca(2+) signaling. This feature, together with previous work in our laboratory, has suggested that calpain, a Ca(2+)-dependent protease, plays a key role in APP α-processing. In this study we report that overexpression of the μ-calpain gene in HEK293 cells resulted in a 2.7-fold increase of the protein levels. Measurements of intracellular calpain enzymatic activity revealed that the calpain overexpressing cells displayed a prominent elevation of the activity compared to wild-type cells. When the cells were stimulated by nicotine, glutamate or phorbol 12,13-dibutylester, the activity increase was even more remarkable and sensitive to calpeptin, a calpain inhibitor. Meanwhile, APP secretion from the calpain overexpressing cells was robustly increased under both resting and stimulated conditions over wild-type cells. Furthermore, cell surface biotinylation experiments showed that μ-calpain was clearly detected among the cell surface proteins. These data together support our view that calpain should be a reasonable candidate for α-secretase for further study. This model is discussed with an interesting fact that three other deposited proteins (tau, spectrin and crystalline) are also the known substrates of calpain. Finally we discuss some current misconceptions in senile dementia research.

  18. High-Throughput, Automated Protein A Purification Platform with Multiattribute LC-MS Analysis for Advanced Cell Culture Process Monitoring. (United States)

    Dong, Jia; Migliore, Nicole; Mehrman, Steven J; Cunningham, John; Lewis, Michael J; Hu, Ping


    The levels of many product related variants observed during the production of monoclonal antibodies are dependent on control of the manufacturing process, especially the cell culture process. However, it is difficult to characterize samples pulled from the bioreactor due to the low levels of product during the early stages of the process and the high levels of interfering reagents. Furthermore, analytical results are often not available for several days, which slows the process development cycle and prevents "real time" adjustments to the manufacturing process. To reduce the delay and enhance our ability to achieve quality targets, we have developed a low-volume, high-throughput, and high-content analytical platform for at-line product quality analysis. This workflow includes an automated, 96-well plate protein A purification step to isolate antibody product from the cell culture fermentation broth, followed by rapid, multiattribute LC-MS analysis. We have demonstrated quantitative correlations between particular process parameters with the levels of glycosylated and glycated species in a series of small scale experiments, but the platform could be used to monitor other attributes and applied across the biopharmaceutical industry.

  19. Processing, physico-chemical, sensory and nutritional evaluation of protein, mineral and vitamin enriched peanut chikki - an Indian traditional sweet. (United States)

    Pallavi, Byrappa Vasu; Chetana, Ramakrishna; Reddy, Sunkireddy Yella


    Chikki, a popular Indian traditional sweet snack prepared from peanut was enriched with protein, minerals and vitamins by incorporating soy protein isolate, calcium carbonate, ferrous fumerate, vitamin A and folic acid to meet the growing demand for health foods. The enriched nutra chikki was evaluated for physico-chemical characteristics such as moisture, texture, peroxide value (PV), fatty acid composition in comparison with control peanut chikki. Nutra chikki, which had initial moisture content of 2-3%, did not alter much up to 60 days of storage at 27 °C and 37 °C. PV increased gradually at 27 °C, whereas at 37 °C, it was higher. Hardness of nutra chikki did not change significantly when stored at 27 °C up to 105 days, but at 37 °C a gradual increase in hardness was observed after 45 days. These results of nutra chikki were similar and comparable with those of control chikki. Nutra chikki had 18% protein, 20% fat, 6.42% Ca, 1.7% Fe, 4000 μg vitamin A and 2660 μg folic acid. Protein digestibility corrected amino acid score of nutra chikki was 0.78 whereas that of control chikki was 0.73. The fatty acid composition of control and nutra chikki was same as both contain peanuts as oil source. The formulation and process parameters for preparation of protein, mineral and vitamin enriched peanut chikki, were standardized. The storage stability and quality parameters of enriched chikki were comparable with those of control chikki.

  20. Prion protein expression and processing in human mononuclear cells: the impact of the codon 129 prion gene polymorphism.

    Directory of Open Access Journals (Sweden)

    Christiane Segarra

    Full Text Available BACKGROUND: So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD, thought to result from the Bovine Spongiform Encephalopathy (BSE prion agent, have shown Methionine-Methionine (M/M homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs from three blood donor populations with Methionine-Methionine (M/M, Valine-Valine (V/V and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism. CONCLUSIONS/SIGNIFICANCE: These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.

  1. Anchoring of c-myc on nuclear matrix proteins in process of mouse thymic T lymphocyte proliferation induced by ConA

    Institute of Scientific and Technical Information of China (English)

    曾丛梅; 蔡树涛; 周凤兰; 张锦珠; 王平


    Isolation and characteriation of functional nudear matrix proteins involved in DNA anchoring and gene expression is one of the major subjects of current nudear matrix research. Southwestern blotting (DNA-protein hybridization) was applied to studying the anchoring of c-myc on the nudear matrix proteins in mouse thymic T lymphocytes. The results showed that c-myc bound to the lamin, p34 and p36 nudear matrix proteins specifically. In the process of mouse thymic PNA T lymphocytes proliferation induced by ConA, the anchoring of c-myc on p34 and p36 nudear matrix proteins changed dynamically.

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

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    Marcelo Arthur CAVALLI


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

  3. Bioactive proteins and energy value of okara as a byproduct in hydrothermal processing of soy milk. (United States)

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


    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 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.

  4. Optimization of protein extraction process from jackfruit seed flour by reverse micelle system

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    Maycon Fagundes Teixeira Reis


    Full Text Available The extraction of protein from flour of jackfruit seeds by reverse micelles was evaluated. Reverse micelle system was composed of sodium dodecyl sulfate (SDS as surfactant, butanol as solvent, and water. The effects of stirring time, temperature, molar ratio H2O SDS-1, concentration of butanol (mass percentage and flour mass were tested in batch systems. Based on the adjusted linear regression model, only butanol concentration provided optimum extraction conditions (41.16%. Based on the analysis of surface response, the best extraction yield could be obtained at 25°C, stirring time of 120 min, mass of flour of 100 mg, and a ratio H2O SDS-1 of 50. Experimental results showed that a 79.00% extraction yield could be obtained.

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

    DEFF Research Database (Denmark)


    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...

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

    NARCIS (Netherlands)

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


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

  7. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells. (United States)

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


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

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

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    Katja Meyer


    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.

  9. 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. (United States)

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


    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.

  10. DNA Microarray-Based Identification of Genes Controlled by Autoinducer 2-Stimulated Quorum Sensing in Escherichia Coli (United States)


    lipopolysaccharide glucosyltransferase 13.4 rnk Regulator of nucleoside diphosphate kinase 13.1 motB Enables flagellar motor rotation, linking torque...S subunit 25.6 ugpC ATP-binding component of sn-glycerol 3-phosphate transport system 26.2 thiH Thiamin biosynthesis, thiazole moiety 219.2 a Genome

  11. Identification of Important Process Variables for Fiber Spinning of Protein Nanotubes Generated from Waste Materials (United States)


    fibers, identification of important spin dope processing and fiber spinning parameters for further opti Fibers, biopolymer , sustainable without disrupting their integrity 19-23, 27, 30. Due to these properties, TMV and M13 are particularly interesting scaffolds for developing...thinning along the fiber were recorded. Under polarizing light, birefringence, which is used to evaluate molecular alignment of the biopolymer chains

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


    Vonghia, G; Liuzzi, V.; M. Faccia; G. Alviti; G. Cacace; A. Di Luccia


    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, ...

  13. A novel bottom-up process to produce nanoparticles containing protein and peptide for suspension in hydrofluoroalkane propellants. (United States)

    Tan, Yinhe; Yang, Zhiwen; Peng, Xinsheng; Xin, Feng; Xu, Yuehong; Feng, Min; Zhao, Chunshun; Hu, Haiyan; Wu, Chuanbin


    To overcome the disadvantages of microemulsion and nanoprecipitation methods to produce protein-containing nanoparticles, a novel bottom-up process was developed to produce nanoparticles containing the model protein lysozyme. The nanoparticles were generated by freeze-drying a solution of lysozyme, lecithin and lactose in tert-butyl alcohol (TBA)/water co-solvent system and washing off excess lecithin in lyophilizate by centrifugation. Formulation parameters such as lecithin concentration in organic phase, water content in TBA/water co-solvent, and lactose concentration in water were optimized so as to obtain desired nanoparticles with retention of the bioactivity of lysozyme. Based on the results, 24.0% (w/v) of lecithin, 37.5% (v/v) of water content, and 0.56% (w/v) of lactose concentration were selected to generate spherical nanoparticles with approximately 200 nm in mean size, 0.1 in polydispersity index (PI), and 99% retained bioactivity of lysozyme. These nanoparticles rinsed with ethanol containing dipalmitoylphosphatidylcholine (DPPC), Span 85 or oleic acid (3%, w/v) could readily be dispersed in HFA 134a to form a stable suspension with good redispersibility and 98% retained bioactivity of lysozyme. The study indicates there is a potential to produce pressed metered dose inhaler (pMDI) formulations containing therapeutic protein and peptide nanoparticles.

  14. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein. (United States)

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


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

  15. Visualization of amino acid composition differences between processed protein from different animal species by self-organizing feature maps

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    Xingfan ZHOU,Zengling YANG,Longjian CHEN,Lujia HAN


    Full Text Available Amino acids are the dominant organic components of processed animal proteins, however there has been limited investigation of differences in their composition between various protein sources. Information on these differences will not only be helpful for their further utilization but also provide fundamental information for developing species-specific identification methods. In this study, self-organizing feature maps (SOFM were used to visualize amino acid composition of fish meal, and meat and bone meal (MBM produced from poultry, ruminants and swine. SOFM display the similarities and differences in amino acid composition between protein sources and effectively improve data transparency. Amino acid composition was shown to be useful for distinguishing fish meal from MBM due to their large concentration differences between glycine, lysine and proline. However, the amino acid composition of the three MBMs was quite similar. The SOFM results were consistent with those obtained by analysis of variance and principal component analysis but more straightforward. SOFM was shown to have a robust sample linkage capacity and to be able to act as a powerful means to link different sample for further data mining.

  16. Applications of Recombinant DNA Technology in Gastrointestinal Medicine and Hepatology: Basic Paradigms of Molecular Cell Biology. Part C: Protein Synthesis and Post-Translational Processing in Eukaryotic Cells

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    Gary E Wild


    Full Text Available The translation of mRNA constitutes the first step in the synthesis of a functional protein. The polypeptide chain is subsequently folded into the appropriate three-dimensional configuration and undergoes a variety of processing steps before being converted into its active form. These processing steps are intimately related to the cellular events that occur in the endoplasmic reticulum and Golgi compartments, and determine the sorting and transport of different proteins to their appropriate destinations within the cell. While the regulation of gene expression occurs primarily at the level of transcription, the expression of many genes can also be controlled at the level of translation. Most proteins can be regulated in response to extracellular signals. In addition, intracellular protein levels can be controlled by differential rates of protein degradation. Thus, the regulation of both the amounts and activities of intracellular proteins ultimately determines all aspects of cell behaviour.

  17. The byssus of the zebra mussel, Dreissena polymorpha. I: Morphology and in situ protein processing during maturation. (United States)

    Rzepecki, L M; Waite, J H


    The zebra mussel, Dreissena polymorpha, owes its notoriety as a biofouler to its adhesive skills and opportunism. Adhesion by the adult mussel to hard substrata is mediated by a nonliving extracorporeal structure called the byssus, which is superficially similar to the byssus of marine mussels in that it consists of a tight bundle of sclerotized threads tipped by adhesive plaques. Juvenile zebra mussels secrete a homologous structure on settlement, but they also employ an elongated belaying byssus while climbing that consists of an elastic, mucous filament anchored at irregular intervals by a byssal thread and plaque. This multiply anchored belaying line can be 20 to 30 times the mussel length. Histochemical tests show that the thread and plaque of both kinds of byssus contains a complex distribution of proteins that are subject to chemical processing after secretion. This processing may result from the formation of crosslinks following the catecholoxidase-catalyzed oxidation of peptidyl 3,4-dihydroxyphenylalanine during sclerotization.

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


    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

  19. Consequences of Inhibiting Amyloid Precursor Protein Processing Enzymes on Synaptic Function and Plasticity

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    Hui Wang


    Full Text Available Alzheimer's disease (AD is a neurodegenerative disease, one of whose major pathological hallmarks is the accumulation of amyloid plaques comprised of aggregated β-amyloid (Aβ peptides. It is now recognized that soluble Aβ oligomers may lead to synaptic dysfunctions early in AD pathology preceding plaque deposition. Aβ is produced by a sequential cleavage of amyloid precursor protein (APP by the activity of β- and γ-secretases, which have been identified as major candidate therapeutic targets of AD. This paper focuses on how Aβ alters synaptic function and the functional consequences of inhibiting the activity of the two secretases responsible for Aβ generation. Abnormalities in synaptic function resulting from the absence or inhibition of the Aβ-producing enzymes suggest that Aβ itself may have normal physiological functions which are disrupted by abnormal accumulation of Aβ during AD pathology. This interpretation suggests that AD therapeutics targeting the β- and γ-secretases should be developed to restore normal levels of Aβ or combined with measures to circumvent the associated synaptic dysfunction(s in order to have minimal impact on normal synaptic function.

  20. A novel coding method for gene mutation correction during protein translation process. (United States)

    Zhang, Lei; Tian, Fengchun; Wang, Shiyuan; Liu, Xiao


    In gene expression, gene mutations often lead to negative effect of protein translation in prokaryotic organisms. With consideration of the influences produced by gene mutation, a novel method based on error-correction coding theory is proposed for modeling and detection of translation initiation in this paper. In the proposed method, combined with a one-dimensional codebook from block coding, a decoding method based on the minimum hamming distance is designed for analysis of translation efficiency. The results show that the proposed method can recognize the biologically significant regions such as Shine-Dalgarno region within the mRNA leader sequences effectively. Also, a global analysis of single base and multiple bases mutations of the Shine-Dalgarno sequences are established. Compared with other published experimental methods for mutation analysis, the translation initiation can not be disturbed by multiple bases mutations using the proposed method, which shows the effectiveness of this method in improving the translation efficiency and its biological relevance for genetic regulatory system.


    Directory of Open Access Journals (Sweden)

    S. L. Menezes


    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.

  2. Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1. (United States)

    Perttilä, Julia; Spuul, Pirjo; Ahola, Tero


    Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse-chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER-Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.

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

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    Maria Cesarina Abete


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

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

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    Entsar Saheb


    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.

  5. Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.

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    Stuart K Archer


    Full Text Available Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9--LIGKA, PNT1 and PNT2--were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3' UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3' UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.

  6. Processing of the Bovine Spongiform Encephalopathy-Specific Prion Protein by Dendritic Cells (United States)

    Rybner-Barnier, Catherine; Jacquemot, Catherine; Cuche, Céline; Doré, Grégory; Majlessi, Laleh; Gabellec, Marie-Madeleine; Moris, Arnaud; Schwartz, Olivier; Di Santo, James; Cumano, Ana; Leclerc, Claude; Lazarini, Françoise


    Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrPbse) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrPbse and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrPbse within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrPbse is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrPbse when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrPbse capture is probably specific to antigen-presenting cells since no uptake of PrPbse was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrPbse. Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 20/common cytokine γ chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings. PMID:16641258

  7. The Chemical Basis for the Origin of the Genetic Code and the Process of Protein Synthesis (United States)

    Lacey, James C., Jr.


    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.

  8. Processing of the bovine spongiform encephalopathy-specific prion protein by dendritic cells. (United States)

    Rybner-Barnier, Catherine; Jacquemot, Catherine; Cuche, Céline; Doré, Grégory; Majlessi, Laleh; Gabellec, Marie-Madeleine; Moris, Arnaud; Schwartz, Olivier; Di Santo, James; Cumano, Ana; Leclerc, Claude; Lazarini, Françoise


    Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrP(bse)) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrP(bse) and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrP(bse) within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrP(bse) is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrP(bse) when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrP(bse) capture is probably specific to antigen-presenting cells since no uptake of PrP(bse) was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrP(bse). Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 2(0)/common cytokine gamma chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings.

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

    DEFF Research Database (Denmark)

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


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

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

    Directory of Open Access Journals (Sweden)

    Krystyna Szymandera-Buszka


    Full Text Available   Fortification of processed meat with iodised table salt was shown to increase thiamine losses, both during thermal processing and storage. Taking into consideration the fact, as well as the recommendation for reduction of consumption of table salt, alternative iodine carriers need to be searched for. Thus the aim of the study was to determine the effect of soy protein isolate (SPI and wheat fibre (WF as iodine salts’ (potassium iodide and iodate carriers on thiamine stability in selected processed meats (steamed meatballs and burgers. The results were compared to the effect of iodised table salt. The highest thiamine losses were found in the presence of iodised table salt, both in the form of iodide and iodate. The application of iodised WF and SPI significantly limited thiamine losses in the course of steaming. It also made possible to reduce thiamine losses during storage in relation to iodised table salt. It was found that the application of WF and SPI as iodine carriers facilitates increased stability of thiamine in relation to table salt during processing and storage of the meat dishes.  

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

    Directory of Open Access Journals (Sweden)

    Mercedes Campderros


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

  12. Effect of simulated processing on the antioxidant capacity and in vitro protein digestion of fruit juice-milk beverage model systems. (United States)

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


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

  13. Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process


    Welderufael, Fisseha Tesfay; Gibson, Trevor; Methven, Lisa; Jauregi, Paula


    The overall aim of this work was to characterize the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilization of β-lactoglobulin and casein derived peptides (CDP) from whey. The adsorbed proteins were hydrolyzed in-situ which resulted in the separat...

  14. The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system. (United States)

    Rogulja-Ortmann, Ana; Picao-Osorio, Joao; Villava, Casandra; Patraquim, Pedro; Lafuente, Elvira; Aspden, Julie; Thomsen, Stefan; Technau, Gerhard M; Alonso, Claudio R


    The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

  15. Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis. (United States)

    Köster, Tino; Meyer, Katja; Weinholdt, Claus; Smith, Lisa M; Lummer, Martina; Speth, Corinna; Grosse, Ivo; Weigel, Detlef; Staiger, Dorothee


    The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

  16. 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: [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)


    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.

  17. Influence of the timing of switching a protein-free to a protein-containing diet on the wound healing process in a rat all-layer skin defect. (United States)

    Tsuda, Kyoko; Nakatani, Toshio; Sugama, Junko; Okuwa, Mayumi; Sanada, Hiromi


    We prepared full thickness skin defects in rats fed on a protein-free diet as a hypoproteinaemia model, then switched the animals to a diet containing a normal protein level 1, 6 or 12 days after wounding (inflammatory, granulation and rearrangement phases of the wound healing process) to examine whether improvement in the low-protein state promotes subsequent wound healing. The interval until wound healing in rats fed on a normal protein diet was significantly shorter, whereas that in rats continuously fed on a protein-free diet was significantly longer than those of other groups. Early correction tended to accelerate wound healing. Although wound contraction in groups receiving a protein-corrected or protein-free diet remained similar until 15 days after wounding, thereafter the duration of the rearrangement phase was significantly longer in the protein-free group than in the other groups. The collagen level per unit of granulation tissue area during wound healing was significantly lower in the protein-free group than in the other groups. These findings indicate that protein correction at any time after wounding accelerates wound healing, although early correction is more effective, and reduces the duration of the rearrangement phase more than those of the inflammatory and granulation phases because of the deposit of collagen.

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

    Directory of Open Access Journals (Sweden)

    Mar Ruperto


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

  19. Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10. (United States)

    Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Benjannet, Suzanne; Chamberland, Ann; Day, Robert; Szumska, Dorota; Constam, Daniel; Bhattacharya, Shoumo; Prat, Annik; Seidah, Nabil G


    Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.

  20. Furin Is the Major Processing Enzyme of the Cardiac-specific Growth Factor Bone Morphogenetic Protein 10* (United States)

    Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Benjannet, Suzanne; Chamberland, Ann; Day, Robert; Szumska, Dorota; Constam, Daniel; Bhattacharya, Shoumo; Prat, Annik; Seidah, Nabil G.


    Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (∼60 kDa) is processed into active BMP10 (∼14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR316↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10. PMID:21550985

  1. Recovery of residual soluble protein by two-step precipitation process with concomitant COD reduction from the yeast-cultivated cheese whey. (United States)

    Yadav, J S S; Yan, S; More, T T; Tyagi, R D; Surampalli, R Y


    The present study was conducted to recover the residual soluble protein after cultivation of yeast (K. marxianus) in cheese whey. Cheese whey continuous fermentation with cell recycle system was carried out at 40 °C and pH 3.5. The yeast biomass was separated from the fermented broth by centrifugation and residual soluble protein from fermented whey supernatant was precipitated by heat treatment (at 100 °C, pH 4.5 and 10 min incubation). The maximum soluble protein recovery up to 53 % was achieved at pH 4.5 with 54 % residual COD removal. However, gravity sedimentable precipitates were obtained at pH 3.5 with 47 % protein recovery. Therefore, the reactor (scale up) study was conducted at pH 3.5 with agitation, which resulted in 68 % of residual soluble protein recovery and simultaneously residual COD removal of 62 %. Further precipitation/coagulation of soluble protein was also evaluated using carboxymethylcellulose (CMC) and then two precipitation (thermal followed by CMC precipitation) processes were combined to increase the protein precipitation, which finally reached up to 81 % of total soluble protein recovery from the supernatant. This optimized process could be applied to recover the residual protein left after fermentation of cheese whey without centrifugation.

  2. 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:, E-mail:, E-mail: [Division of Bioengineering, National University of Singapore (Singapore)


    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.

  3. Activator of G-protein signaling 8 is involved in VEGF-mediated signal processing during angiogenesis. (United States)

    Hayashi, Hisaki; Al Mamun, Abdullah; Sakima, Miho; Sato, Motohiko


    Activator of G-protein signaling 8 (AGS8, also known as FNDC1) is a receptor-independent accessory protein for the Gβγ subunit, which was isolated from rat heart subjected to repetitive transient ischemia with the substantial development of collaterals. Here, we report the role of AGS8 in vessel formation by endothelial cells. Knockdown of AGS8 by small interfering RNA (siRNA) inhibited vascular endothelial growth factor (VEGF)-induced tube formation, as well as VEGF-stimulated cell growth and migration. VEGF stimulated the phosphorylation of the VEGF receptor-2 (VEGFR-2, also known as KDR), ERK1/2 and p38 MAPK; however, knockdown of AGS8 inhibited these signaling events. Signal alterations by AGS8 siRNA were associated with a decrease of cell surface VEGFR-2 and an increase of VEGFR-2 in the cytosol. Endocytosis blockers did not influence the decrease of VEGFR-2 by AGS8 siRNA, suggesting the involvement of AGS8 in VEGFR-2 trafficking to the plasma membrane. VEGFR-2 formed a complex with AGS8 in cells, and a peptide designed to disrupt AGS8-Gβγ interaction inhibited VEGF-induced tube formation. These data suggest a potential role for AGS8-Gβγ in VEGF signal processing. AGS8 might play a key role in tissue adaptation by regulating angiogenic events.

  4. Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jinjin; Xing, Xu; Herr, Andrew B.; Bell, Charles E.; (OSU); (UCIN)


    Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5{prime}-ended strand to form 5{prime}-mononucleotides and a 3{prime}-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 {angstrom} resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 {angstrom} from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5{prime}-ended strand passes through a tunnel to access one of the four active sites, and the 3{prime}-ended strand passes through the plugged end of the channel at the back of the tetramer.

  5. 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.

  6. 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.

  7. Effects of different industrial heating processes of milk on site-specific protein modifications and their relationship to in vitro and in vivo digestibility. (United States)

    Wada, Yasuaki; Lönnerdal, Bo


    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.

  8. 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


    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.

  9. Changes in protein structures to improve the rheology and texture of reduced-fat sausages using high pressure processing. (United States)

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


    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.

  10. In vitro digestibility, protein composition and techno-functional properties of Saskatchewan grown yellow field peas (Pisum sativum L.) as affected by processing. (United States)

    Ma, Zhen; Boye, Joyce I; Hu, Xinzhong


    Saskatchewan grown yellow field pea was subjected to different processing conditions including dehulling, micronization, roasting, conventional/microwave cooking, germination, and combined germination and conventional cooking/roasting. Their nutritional and antinutritional compositions, functional properties, microstructure, thermal properties, in vitro protein and starch digestibility, and protein composition were studied. Processed field peas including conventional cooked yellow peas (CCYP), microwave cooked yellow peas (MCYP), germinated-conventional cooked yellow peas (GCCYP), and germinated-roasted yellow peas (GRYP) exhibited the significantly higher in vitro protein digestibility (IVPD), which was in accordance with their significantly lower trypsin inhibitor activity and tannin content. The SDS-PAGE and size exclusion HPLC profiles of untreated pea proteins and their hydrolysates also confirmed the IVPD result that these four treatments facilitated the hydrolysis of pea proteins to a greater extent. The CCYP, MCYP, GCCYP, and GRYP also exhibited significantly higher starch digestibility which was supported by their lower onset (To), peak (Tp), and conclusion (Tc) temperatures obtained from DSC thermogram, their lower pasting properties and starch damage results, as well as their distinguished amorphous flakes' configuration observed on the scanning electron microscopic image. LC/ESI-MS/MS analysis following in-gel digests of SDS-PAGE separated proteins allowed detailed compositional characterization of pea proteins. The present study would provide fundamental information to help to better understand the functionality of field peas as ingredients, and particularly in regards to agri-food industry to improve the process efficiency of field peas with enhanced nutritional and techno-functional qualities.

  11. Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process. (United States)

    Hapuarachchi, Suminda; Fodor, Szilan; Apostol, Izydor; Huang, Gang


    Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.

  12. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham. (United States)

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


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

  13. Effects of processing moisture on the physical properties and in vitro digestibility of starch and protein in extruded brown rice and pinto bean composite flours. (United States)

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


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

  14. Effects of ethanol on aggregation, serotonin release, and amyloid precursor protein processing in rat and human platelets. (United States)

    Ehrlich, Daniela; Humpel, Christian


    It is known that oxidative stress leads to amyloid precursor protein (APP) dysregulation in platelets. Ethanol (EtOH) is a vascular risk factor and induces oxidative stress. The aim of the present study was thus to investigate whether EtOH affects APP processing in rat and human platelets. Platelets were exposed to 50 mM EtOH with and without 2 mM calcium-chloride (CaCl₂) for 20 or 180 minutes at 37°C. Platelet aggregation, serotonin release and APP isoforms 130 and 106/110 kDa were analyzed. As a control, 100 mM H₂O₂ was tested in rat platelets. Our data show that EtOH alone did not affect any of the analyzed parameters, whereas CaCl₂ significantly increased aggregation of rat and human platelets. In addition, CaCl₂ alone enhanced serotonin release in rat platelets. EtOH counteracted CaCl₂-induced aggregation and serotonin release. In the presence of CaCl₂, EtOH reduced the 130 kDa APP isoform in rat and human platelets. In conclusion, this study shows that in the presence of CaCl₂, EtOH affects the platelet function and APP processing in rat and human platelets.

  15. 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.

  16. Molecular spectroscopic investigation on fractionation-induced changes on biomacromolecule of co-products from bioethanol processing to explore protein metabolism in ruminants (United States)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gemelli, Claudia, E-mail: [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)


    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.

  18. Studies on the development of infant foods from plant protein sources. Part II. Effect of processing conditions on the chemical and nutritive properties of chickpea (Cicer arietinum). (United States)

    Khaleque, A; Elías, L G; Braham, J E; Bressani, R


    In order to improve the taste, flavor and nutritional quality of chickpea (Cicer arietinum), various processing conditions were studied. The decorticated samples were processed under various conditions, either by presoaking or non-soaking in water or sodium carbonate solution. The proteins were also isolated from water or carbonate-presoaked chickpea and subjected to various processing. Carbonate-presoaked samples gave slightly lower protein and ash values. No major changes in other constituents were observed. Subjective analysis of the intensity of characteristic chickpea flavor in processed samples was carried out, indicating some improvement in the carbonate-presoaked samples. Carbonate-treated samples exhibited a lighter color. The carbonate presoaking procedure had no adverse effect on the availability of lysine and nitrogen solubility index (NSI), as compared to the water-presoaking procedure. The time required to inactivate trypsin inhibitors in carbonate-presoaked chickpea at boiling temperature, was half that required in the case of water-presoaked ones. Under the conditions used in treating chickpea with sodium carbonate, no beneficial effect was observed in reducing the tannin content. No significant differences were observed in net protein ratio (NPR) among the various processed chickpea samples, even though in some cases isolated protein gave significantly lower NPR values. Digestibility values were higher for isolated protein than for whole chickpea samples.

  19. Multi-dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

    NARCIS (Netherlands)

    Ahamed, T.; Pinkse, M.W.H.; Wielen, van der L.A.M.; Verhaert, P.; Dedem, G.; Eppink, M.H.M.; Sandt, van de E.; Ottens, M.


    A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and size-exclusion chromato

  20. Intravirion Processing of the Human Immunodeficiency Virus Type 1 Vif Protein by the Viral Protease May Be Correlated with Vif Function


    Khan, Mohammad A.; Akari, Hirofumi; Kao, Sandra; Aberham, Claudia; Davis, David; Buckler-White, Alicia; Strebel, Klaus


    The human immunodeficiency virus type 1 (HIV-1) Vif protein is specifically packaged into virus particles through an interaction with viral genomic RNA in which it associates with the viral nucleoprotein complex. We now demonstrate for the first time that virus-associated Vif is subject to proteolytic processing by the viral protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type independent and evident by the appearance of...

  1. The purinergic receptor P2X7 triggers alpha-secretase-dependent processing of the amyloid precursor protein. (United States)

    Delarasse, Cécile; Auger, Rodolphe; Gonnord, Pauline; Fontaine, Bertrand; Kanellopoulos, Jean M


    The amyloid precursor protein (APP) is cleaved by β- and γ-secretases to generate the β-amyloid (Aβ) peptides, which are present in large amounts in the amyloid plaques of Alzheimer disease (AD) patient brains. Non-amyloidogenic processing of APP by α-secretases leads to proteolytic cleavage within the Aβ peptide sequence and shedding of the soluble APP ectodomain (sAPPα), which has been reported to be endowed with neuroprotective properties. In this work, we have shown that activation of the purinergic receptor P2X7 (P2X7R) stimulates sAPPα release from mouse neuroblastoma cells expressing human APP, from human neuroblastoma cells and from mouse primary astrocytes or neural progenitor cells. sAPPα shedding is inhibited by P2X7R antagonists or knockdown of P2X7R with specific small interfering RNA (siRNA) and is not observed in neural cells from P2X7R-deficient mice. P2X7R-dependent APP-cleavage is independent of extracellular calcium and strongly inhibited by hydroxamate-based metalloprotease inhibitors, TAPI-2 and GM6001. However, knockdown of a disintegrin and metalloproteinase-9 (ADAM9), ADAM10 and ADAM17 by specific siRNA, known to have α-secretase activity, does not block the P2X7R-dependent non-amyloidogenic pathway. Using several specific pharmacological inhibitors, we demonstrate that the mitogen-activated protein kinase modules Erk1/2 and JNK are involved in P2X7R-dependent α-secretase activity. Our study suggests that P2X7R, which is expressed in hippocampal neurons and glial cells, is a potential therapeutic target in AD.

  2. 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;


    Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent ......Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for c......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...

  3. 玉米蛋白加工利用研究进展%Advance of processing and application of corn protein

    Institute of Scientific and Technical Information of China (English)

    敬珊珊; 刘晓兰; 郑喜群


    In corn wet milling, corn proteins exist in corn gluten meal, com germ meal and corn steep liquor. Characteristics of corn protein, such as amino acid composition, processing condition and processing functionality of corn protein were reviewed. Applying potential of corn protein hydrolysate in food industry was pointed out.%在湿法玉米淀粉加工中,玉米蛋白存在于副产物玉米黄粉、玉米胚芽饼和玉米浆中.文章在分析玉米蛋白氨基酸组成等特性的基础上,综合分析玉米蛋白加工利用现状和玉米蛋白水解物的功能特性,指出玉米蛋白水解物在食品工业上应用的潜力.

  4. Label-free, real-time detection of the dynamic processes of protein degradation using oblique-incidence reflectivity difference method (United States)

    Liu, S.; Zhu, J. H.; He, L. P.; Dai, J.; Lu, H. B.; Wu, L.; Jin, K. J.; Yang, G. Z.; Zhu, H.


    Based on the requirements for studying the dynamic process of proteinase action substrates in life science, we selected six random proteins including 1L-10, SCGB2A2, CENPQ, GST, HK1, KLHL7, as well as five different concentrations of 1L-10 proteins of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.0625 mg/ml, and fabricated two types of substrate protein microarrays, respectively. We detected the dynamic processes of proteins degraded by proteinase K using oblique-incidence reflectivity difference (OIRD) method in a label-free and real-time manner. We obtained the relevant degradation velocities and the degradation time. The experimental results demonstrate that OIRD has the ability to study proteinase action substrates which is out of reach of label methods and is expected to offer opportunities to determine protease-substrate relationships on the systems biology level.

  5. 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: [University of Bremen, Advanced Ceramics, Am Biologischen Garten 2, 28359 Bremen (Germany); Koch, Dietmar, E-mail: [German Aerospace Center, Ceramic Composite Structures, Pfaffenwaldring 38-40, 70569 Stuttgart (Germany); Lutz, Rainer, E-mail: [University of Erlangen-Nuremberg, Department of Oral and Maxillofacial Surgery, Glueckstrasse 11, 91054 Erlangen (Germany); Schlegel, Karl A., E-mail: [University of Erlangen-Nuremberg, Department of Oral and Maxillofacial Surgery, Glueckstrasse 11, 91054 Erlangen (Germany); Treccani, Laura, E-mail: [University of Bremen, Advanced Ceramics, Am Biologischen Garten 2, 28359 Bremen (Germany); Rezwan, Kurosch, E-mail: [University of Bremen, Advanced Ceramics, Am Biologischen Garten 2, 28359 Bremen (Germany)


    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.


    Directory of Open Access Journals (Sweden)

    O. Shalimova


    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.

  7. DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation. (United States)

    Burger, Amanda; Raymer, Jenny; Bockrath, R


    UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

  8. 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


    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.

  9. [Effect of the extrusion process on the functional characteristics and protein quality of quinua (Chenopodium quinoa, Willd)]. (United States)

    Romero, A; Bacigalupo, A; Bressani, R


    In order to have available a human food of high nutritive value, and conscious of the protein quality of the quinua, as well as its carbohydrate, vitamin and mineral content, its behavior during the extrusion process was tested in the present study. To eliminate saponins, a simple method was developed which consisted of washing the seeds through an aluminum container, using a wooden stirrer. Seven treatments were studied: washed quinua, washed and cooked quinua, washed and expanded quinua No. 1 and No. 2, and washed and texturized quinua No. 1 and No. 2; casein was used as control. Biological evaluation trials were carried out in Holtzman rats, following the PER method. To detect the possible effects of the processed quinua on the experimental animals, hematological as well as histopathological studies of the vital organs were performed. A maximum PER of 2.43 was obtained for the texturized quinua, 2.16 for the expanded quinua, 2.6 for the cooked quinua, while the casein control yielded a PER of 3.00. The physico-chemical characteristics of the quinua flour were determined, as well as those of the expanded and texturized products. The product obtained was subjected to an organoleptic trial and it can be stated that the results obtained were satisfactory. The product can be consumed directly without major modifications and has an acceptable flavor. The nutritive value of quinua was not impaired; it compared favorably with the best diets recommended for the population, especially of those with a lower income. The results obtained in the present study suggest the possibility of increasing the nutritional value of the product, as well as its acceptability.

  10. Proteomic and transcriptomic analysis of Arabidopsis seeds: molecular evidence for successive processing of seed proteins and its implication in the stress response to sulfur nutrition. (United States)

    Higashi, Yasuhiro; Hirai, Masami Yokota; Fujiwara, Toru; Naito, Satoshi; Noji, Masaaki; Saito, Kazuki


    Seed storage proteins are synthesized as sources of carbon, nitrogen and sulfur for the next generation of plants. Their composition changes according to nutritional conditions. Here, we report the precise molecular identification of seed proteins by proteomic analysis of wild-type Arabidopsis thaliana and methionine-over-accumulating mutant mto1-1 plants. The identities of 50 protein spots were determined in the protein extract of mature Arabidopsis seeds by two-dimensional (2D) gel electrophoresis and subsequent mass spectrometric analysis. Of these protein spots, 42 were identified as derived from 12S globulins or 2S albumins. These results indicate that approximately 84% of protein species in Arabidopsis seeds are derived from a few genes coding for 12S globulins and 2S albumins. Extensive mass spectrometric analysis of the 42 spots revealed that successive C-terminal degradation occurred on the 12S globulins. The feasibility of this C-terminal processing was rationalized by molecular modeling of the three-dimensional structure of 12S globulins. The C-terminal degradation at glutamic acid residues of the 12S globulin subunits was repressed under sulfur-deficient conditions. Transcriptome analysis was combined with proteomic analysis to elucidate the mechanism of changes in seed protein composition in response to sulfur deficiency. The results suggest that seed storage proteins in Arabidopsis undergo multi-layer regulation, with emphasis on post-translational modifications that enable the plant to respond to sulfur deficiency.

  11. Interaction of arginine with protein during refolding process probed by amide H/D exchange mass spectrometry and isothermal titration calorimetry. (United States)

    Zhao, Dawei; Liu, Yongdong; Zhang, Guifeng; Zhang, Chun; Li, Xiunan; Wang, Qingqing; Shi, Hong; Su, Zhiguo


    Arginine has been widely used as low molecular weight additive to promote protein refolding by suppressing aggregate formation. However, methods to investigate the role of arginine in protein refolding are often limited on protein's global conformational properties. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to study the effects of arginine on recombinant human granulocyte colony-stimulating factor (rhG-CSF) refolding at the scale of peptide mapping. It was found that deuteration levels of rhG-CSF refolded with arginine was higher than that without arginine during the whole refolding process, but they became almost the same when the refolding reached equilibrium. This phenomenon indicated that arginine could protect some amide deuterium atoms from being exchanged with hydrogen, but the protection diminished gradually along with refolding proceeding. Enzymatic digestion revealed six particular peptides of 16-47, 72-84, 84-93, 114-124, 145-153 and 154-162 were mainly responsible for the deuteration, and all of them dominantly located in protein's α-helix domain. Furthermore, thermodynamics analysis by isothermal titration calorimetry provided direct evidence that arginine could only react with denatured and partially refolded rhG-CSF. Taking all of the results together, we suggest that arginine suppresses protein aggregation by a reversible combination. At the initial refolding stage, arginine could combine with the denatured protein mainly through hydrogen bonding. Subsequently, arginine is gradually excluded from protein with protein's native conformation recovering.

  12. Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8.

    Directory of Open Access Journals (Sweden)

    Rachel Senturia

    Full Text Available Human DiGeorge Critical Region 8 (DGCR8 is an essential microRNA (miRNA processing factor that is activated via direct interaction with Fe(III heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD. We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star also binds heme. The extinction coefficients (ε of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

  13. Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

    Directory of Open Access Journals (Sweden)

    Viviana A Cavieres

    Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706 prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1, a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD. Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP, resulting in the accumulation of immature APP (iAPP in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.

  14. Short communication: Proteins in heat-processed skim milk powder have no positive effects on bone loss of ovariectomized rats. (United States)

    Du, M; Kong, Y; Wang, C; Gao, H; Han, X; Yi, H; Zhang, L


    Milk has positive effects on bone growth. However, the effect of skim milk powder (SMP) on bone properties has not been reported. This study investigated the effect of SMP on bone loss in ovariectomized (OVX) rats. Forty female Sprague-Dawley rats were ovariectomized and another 10 rats received a sham operation. The OVX rats were randomly separated into 4 groups: OVX control, OVX SMP1 (SMP at 0.04 g/d), OVX SMP2 (SMP at 0.20 g/d), and OVX SMP3 (SMP at 0.40 g/d). Skim milk powder was supplied in the rat diet for 12 wk, and the rats were gavaged once per day. The effects of SMP on calcium content and bone mineral density of femur were determined by atomic absorption spectrophotometry and dual-energy x-ray absorptiometry, respectively. Compared with the control, SMP at all dose levels tested had no particular effect on weight:length, calcium content, or bone mineral density of femurs. It was demonstrated that SMP (0.04 to 0.40 g/d) had no positive effect on bone loss in OVX rats, probably because the heat treatment used during SMP processing caused a loss of biological activity in the protein.

  15. Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy (United States)

    Muñoz, Vanessa C.; Yefi, Claudia P.; Bustamante, Hianara A.; Barraza, Rafael R.; Tapia-Rojas, Cheril; Otth, Carola; Barrera, María José; González, Carlos; Mardones, Gonzalo A.; Inestrosa, Nibaldo C.; Burgos, Patricia V.


    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo. PMID:26308941

  16. Discrimination of different processed animal proteins (PAPs by FT-IR spectroscopy based on their fat characteristics

    Directory of Open Access Journals (Sweden)

    Pu, Q.


    Full Text Available This study was undertaken to explore the potential of FT-IR technique for discriminating different species of processed animal proteins (PAPs based on their fat characteristics. A total of 47 source-reliable PAPs samples containing fish meal, porcine, bovine, ovine and poultry meat and bone meal (MBM were involved in the present study. The results obtained showed that the FT-IR differentiated quite well between the fat derived from different species of PAPs, especially at the bands of 3,006 cm-1 and 722 cm-1. Results provided evidence that FT-IR differentiated the fat derived from fish meal, terrestrial non-ruminant and ruminant MBM quite well. Fish meal and ruminant MBM samples could be discriminated effectively by both the sensitivity and specificity values which were 1.00 and 1.00, respectively. For non-ruminant MBM samples, the sensitivity and specificity were 1.00 and 0.96, respectively. However, it was hard to distinguish bovine MBM from ovine ones as well as porcine MBM from poultry ones. The result makes it possible to allow FT-IR analytical methodology as a preliminary study for the exploitation of a rapid and reliable way for the identification of the animal origin of PAPs used in feeding stuffs.

  17. The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence. (United States)

    Scarafoni, Alessio; Ronchi, Alessandro; Prinsi, Bhakti; Espen, Luca; Assante, Gemma; Venturini, Giovanni; Duranti, Marcello


    The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

  18. Traceability of processed animal proteins with varying texture in feed: determination with microscopic and polymerase Chain Reaction methods

    Directory of Open Access Journals (Sweden)

    Hormisch D.E.


    Full Text Available To test the traceability of different animal components that could enter the feed chain two methods for the determination of processed animal proteins (PAPs in feed – classical microscopy and polymerase chain reaction (PCR-analysis – were applied in the following study. To determine PAPs of varying but defined structure different animal meals were produced artificially and analysed after spiking to a set of 13 compound feed samples. The aims of the study were (i to compare the capacity and the limits of both methods with respect to the determination of animal constituents of varying composition, (ii to verify a correct interpretation of the results from each method and (iii to determine an optimum application area for each method. Both methods complemented each other. The microscopic approach allowed a reproducible, high sensitive and quantitative determination of animal ingredients with morphological detectable structures, and in the presence of bone fragments a d i fferentiation between fish and terrestrial animals was possible simultaneously. The PCR-analysis provided the detection of animal ingredients in feed even in absence of visible structures but fishmeal was not detected in a sufficient manner by the chosen screening setup. However, the PCR-method enabled to differentiate between animal groups or species and to identify animal species. The methods complemented each other not only in the analytical features but also regarding the results produced by the detection of two different analytical targets of PAPs, morphological structures and gene sequences, r e s p e c t i v e l y. Suitable data regarding the presence of their analytical targets were produced by each method, but a combination of both methods enabled furthermore to report correct results regarding the presence of the artificially composed PAPs in the feed samples. It was concluded that a combination of microscopy and PCR-analysis is reasonable for special application

  19. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes


    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin


    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disord...

  20. A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography. (United States)

    Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen


    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.

  1. The forward and backward transport processes in the AOT/hexane reversed micellar extraction of soybean protein. (United States)

    Chen, Jun; Chen, Fengliang; Wang, Xianchang; Zhao, Xiaoyan; Ao, Qiang


    Soybean protein was taken as a model protein to investigate two aspects of the protein extraction by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles: (1) the forward protein extraction from the solid state, and the effect of pH, AOT concentration, alcohol and water content (W0) on the transfer efficiency; (2) the back-transfer, the capability of the protein to be recovered from the micellar solution. The experimental results led to the conclusion that the highest forward extraction efficiency of soybean protein was reached at AOT concentration 180 mmol l(-1), aqueous pH 7.0, KCl concentration 0.05 mol l(-1), 0.5 % (v/v) alcohol, W0 18. Under these conditions, the forward extraction efficiency of soybean protein achieved 70.1 %. It was noted that the percentage of protein back extraction depended on the salt concentration and pH value. Around 92 % of protein recovery was obtained after back extraction.

  2. Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC. (United States)

    Liu, Wen-Jun; Wu, Qian; Xu, Bi; Zhang, Xin-Yu; Xia, Xiao-Li; Sun, Huai-Chang


    Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human β defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

  3. Sesamin increases heme oxygenase-1 protein in RAW 264.7 macrophages through inhibiting its ubiquitination process. (United States)

    Fukunaga, Mizuki; Ohnishi, Masatoshi; Shiratsuchi, Ayano; Kawakami, Takuya; Takahashi, Madoka; Motomura, Misato; Egusa, Kyohei; Urasaki, Tomoka; Inoue, Atsuko


    Sesamin is a major component in lignans of sesame seed oil, known to possess potent anti-oxidative capacity. In this study, the variation of heme oxygenase (HO)-1, a kind of anti-oxidative enzyme, by sesamin in murine macrophage cell line RAW 264.7 cells was investigated. Lipopolysaccharide (LPS; 10μg/ml) exposure tended to increase HO-1 protein expression. Co-treatment with 100μM sesamin for 12h up-regulated the HO-1 protein level increased by LPS; however, HO-1 mRNA was unaffected. Sesamin delayed the reversal, by the protein synthesis inhibitor cycloheximide (1μM), of the LPS-induced increase of HO-1 protein level. Meanwhile, sesamin suppressed LPS-induced expression of inducible nitric oxide (NO) synthase (iNOS) protein and associated NO release. LPS-induced increase of iNOS protein expression was also reversed by cycloheximide, which was not affected by sesamin, unlike HO-1. To clarify the mechanisms that underlie the up-regulation of HO-1 protein level by sesamin, the human embryonic kidney (HEK) 293T cell line transfected with Flag-tagged HO-1 was used. A proteasome inhibitor, MG-132 (10μM), stabilized HO-1 protein in HEK 293T cells. Co-treatment with sesamin decreased ubiquitinated HO-1 protein accumulation by MG-132. However, sesamin did not affect the proteasome activity. These findings suggest that sesamin disturbs the degradation of HO-1 protein through inhibiting its ubiquitination, resulting in HO-1 protein up-regulation.

  4. Hydrodynamical properties of recombinant spider silk proteins: Effects of pH, salts and shear, and implications for the spinning process. (United States)

    Leclerc, Jérémie; Lefèvre, Thierry; Gauthier, Martin; Gagné, Stéphane M; Auger, Michèle


    We have investigated the effect of pH, salts and shear on the hydrodynamical diameter of recombinant major ampullate (MA) rMaSpI silk proteins in solution as a function of time using (1) H solution NMR spectroscopy. The results indicate that the silk proteins in solution are composed of two diffusing populations, a high proportion of "native" solubilized proteins and a small amount of high molecular weight oligomers. Similar results are observed with the MA gland content. Salts help maintaining the proteins in a compact form in solution over time and inhibit aggregation, the absence of salts triggering protein assembly leading to a gel state. Moreover, the aggregation kinetics of rMaSpI at low salt concentration accelerates as the pH is close to the isoelectric point of the proteins, suggesting that the pH decrease tends to slow down aggregation. The data also support the strong impact of shear on the spinning process and suggest that the assembly is driven by a nucleation conformational conversion mechanism. Thus, the adjustment of the physicochemical conditions in the ampulla seems to promote a stable, long term storage. In addition, the optimization of protein conformation as well as their unfolding and aggregation propensity in the duct leads to a specifically organized structure.

  5. Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder? (United States)

    Schubert, K O; Föcking, M; Prehn, J H M; Cotter, D R


    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.

  6. Global analysis of lysine acetylation suggests the involvement of protein acetylation in diverse biological processes in rice (Oryza sativa.

    Directory of Open Access Journals (Sweden)

    Babi Ramesh Reddy Nallamilli

    Full Text Available Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa. We identified 60 lysine acetylated sites on 44 proteins of diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated non-histone proteins. Examination of the amino acid composition revealed substantial amino acid bias around the acetylation sites and the amino acid preference is conserved among different organisms. Gene ontology analysis demonstrates that lysine acetylation occurs in diverse cytoplasmic, chloroplast and mitochondrial proteins in addition to the histone modifications. Our results suggest that lysine acetylation might constitute a regulatory mechanism for many proteins, including both histones and non-histone proteins of diverse biological functions.

  7. Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process. (United States)

    Cobaleda, C; Muñoz-Barroso, I; Sagrera, A; Villar, E


    Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.

  8. Comparative study of denaturation of whey protein isolate (WPI) in convective air drying and isothermal heat treatment processes. (United States)

    Haque, M Amdadul; Aldred, Peter; Chen, Jie; Barrow, Colin J; Adhikari, Benu


    The extent and nature of denaturation of whey protein isolate (WPI) in convective air drying environments was measured and analysed using single droplet drying. A custom-built, single droplet drying instrument was used for this purpose. Single droplets having 5±0.1μl volume (initial droplet diameter 1.5±0.1mm) containing 10% (w/v) WPI were dried at air temperatures of 45, 65 and 80°C for 600s at constant air velocity of 0.5m/s. The extent and nature of denaturation of WPI in isothermal heat treatment processes was measured at 65 and 80°C for 600s and compared with those obtained from convective air drying. The extent of denaturation of WPI in a high hydrostatic pressure environment (600MPa for 600s) was also determined. The results showed that at the end of 600s of convective drying at 65°C the denaturation of WPI was 68.3%, while it was only 10.8% during isothermal heat treatment at the same medium temperature. When the medium temperature was maintained at 80°C, the denaturation loss of WPI was 90.0% and 68.7% during isothermal heat treatment and convective drying, respectively. The bovine serum albumin (BSA) fraction of WPI was found to be more stable in the convective drying conditions than β-lactoglobulin and α-lactalbumin, especially at longer drying times. The extent of denaturation of WPI in convective air drying (65 and 80°C) and isotheral heat treatment (80°C) for 600s was found to be higher than its denaturation in a high hydrostatic pressure environment at ambient temperature (600MPa for 600s).

  9. Evolution of protein N-glycosylation process in Golgi apparatus which shapes diversity of protein N-glycan structures in plants, animals and fungi (United States)

    Wang, Peng; Wang, Hong; Gai, Jiangtao; Tian, Xiaoli; Zhang, Xiaoxiao; Lv, Yongzhi; Jian, Yi


    Protein N-glycosylation (PNG) is crucial for protein folding and enzymatic activities, and has remarkable diversity among eukaryotic species. Little is known of how unique PNG mechanisms arose and evolved in eukaryotes. Here we demonstrate a picture of onset and evolution of PNG components in Golgi apparatus that shaped diversity of eukaryotic protein N-glycan structures, with an emphasis on roles that domain emergence and combination played on PNG evolution. 23 domains were identified from 24 known PNG genes, most of which could be classified into a single clan, indicating a single evolutionary source for the majority of the genes. From 153 species, 4491 sequences containing the domains were retrieved, based on which we analyzed distribution of domains among eukaryotic species. Two domains in GnTV are restricted to specific eukaryotic domains, while 10 domains distribute not only in species where certain unique PNG reactions occur and thus genes harboring these domains are supoosed to be present, but in other ehkaryotic lineages. Notably, two domains harbored by β-1,3 galactosyltransferase, an essential enzyme in forming plant-specific Lea structure, were present in separated genes in fungi and animals, suggesting its emergence as a result of domain shuffling. PMID:28074929

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

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


    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.

  11. 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. (United States)

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


    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.

  12. A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment. (United States)

    Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C


    Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers

  13. In situ protein secondary structure determination in ice: Raman spectroscopy-based process analytical tool for frozen storage of biopharmaceuticals. (United States)

    Roessl, Ulrich; Leitgeb, Stefan; Pieters, Sigrid; De Beer, Thomas; Nidetzky, Bernd


    A Raman spectroscopy-based method for in situ monitoring of secondary structural composition of proteins during frozen and thawed storage was developed. A set of reference proteins with different α-helix and β-sheet compositions was used for calibration and validation in a chemometric approach. Reference secondary structures were quantified with circular dichroism spectroscopy in the liquid state. Partial least squares regression models were established that enable estimation of secondary structure content from Raman spectra. Quantitative secondary structure determination in ice was accomplished for the first time and correlation with existing (qualitative) protein structural data from the frozen state was achieved. The method can be used in the presence of common stabilizing agents and is applicable in an industrial freezer setup. Raman spectroscopy represents a powerful, noninvasive, and flexibly applicable tool for protein stability monitoring during frozen storage.

  14. 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. (United States)

    Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao


    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.

  15. Stabilization of water in oil in water (W/O/W) emulsion using whey protein isolate-conjugated durian seed gum: enhancement of interfacial activity through conjugation process. (United States)

    Tabatabaee Amid, Bahareh; Mirhosseini, Hamed


    The present work was conducted to investigate the effect of purification and conjugation processes on functional properties of durian seed gum (DSG) used for stabilization of water in oil in water (W/O/W) emulsion. Whey protein isolate (WPI) was conjugated to durian seed gum through the covalent linkage. In order to prepare WPI-DSG conjugate, covalent linkage of whey protein isolate to durian seed gum was obtained by Maillard reaction induced by heating at 60 °C and 80% (±1%) relative humidity. SDS-polyacrylamide gel electrophoresis was used to test the formation of the covalent linkage between whey protein isolate and durian seed gum after conjugation process. In this study, W/O/W stabilized by WPI-conjugated DSG A showed the highest interface activity and lowest creaming layer among all prepared emulsions. This indicated that the partial conjugation of WPI to DSG significantly improved its functional characteristics in W/O/W emulsion. The addition of WPI-conjugated DSG to W/O/W emulsion increased the viscosity more than non-conjugated durian seed gum (or control). This might be due to possible increment of the molecular weight after linking the protein fraction to the structure of durian seed gum through the conjugation process.

  16. Application of quality by design principles to the development and technology transfer of a major process improvement for the manufacture of a recombinant protein. (United States)

    Looby, Mairead; Ibarra, Neysi; Pierce, James J; Buckley, Kevin; O'Donovan, Eimear; Heenan, Mary; Moran, Enda; Farid, Suzanne S; Baganz, Frank


    This study describes the application of quality by design (QbD) principles to the development and implementation of a major manufacturing process improvement for a commercially distributed therapeutic protein produced in Chinese hamster ovary cell culture. The intent of this article is to focus on QbD concepts, and provide guidance and understanding on how the various components combine together to deliver a robust process in keeping with the principles of QbD. A fed-batch production culture and a virus inactivation step are described as representative examples of upstream and downstream unit operations that were characterized. A systematic approach incorporating QbD principles was applied to both unit operations, involving risk assessment of potential process failure points, small-scale model qualification, design and execution of experiments, definition of operating parameter ranges and process validation acceptance criteria followed by manufacturing-scale implementation and process validation. Statistical experimental designs were applied to the execution of process characterization studies evaluating the impact of operating parameters on product quality attributes and process performance parameters. Data from process characterization experiments were used to define the proven acceptable range and classification of operating parameters for each unit operation. Analysis of variance and Monte Carlo simulation methods were used to assess the appropriateness of process design spaces. Successful implementation and validation of the process in the manufacturing facility and the subsequent manufacture of hundreds of batches of this therapeutic protein verifies the approaches taken as a suitable model for the development, scale-up and operation of any biopharmaceutical manufacturing process.

  17. Advances in inline quantification of co-eluting proteins in chromatography: Process-data-based model calibration and application towards real-life separation issues. (United States)

    Brestrich, Nina; Sanden, Adrian; Kraft, Axel; McCann, Karl; Bertolini, Joseph; Hubbuch, Jürgen


    Pooling decisions in preparative liquid chromatography for protein purification are usually based on univariate UV absorption measurements that are not able to differentiate between product and co-eluting contaminants. This can result in inconsistent pool purities or yields, if there is a batch-to-batch variability of the feedstock. To overcome this analytical bottleneck, a tool for selective inline quantification of co-eluting model proteins using mid-UV absorption spectra and Partial Least Squares Regression (PLS) was presented in a previous study and applied for real-time pooling decisions. In this paper, a process-data-based method for the PLS model calibration will be introduced that allows the application of the tool towards chromatography steps of real-life processes. The process-data-based calibration method uses recorded inline mid-UV absorption spectra that are correlated with offline fraction analytics to calibrate PLS models. In order to generate average spectra from the inline data, a Visual Basic for Application macro was successfully developed. The process-data-based model calibration was established using a ternary model protein system. Afterwards, it was successfully demonstrated in two case studies that the calibration method is applicable towards real-life separation issues. The calibrated PLS models allowed a successful quantification of the co-eluting species in a cation-exchange-based aggregate and fraction removal during the purification of monoclonal antibodies and of co-eluting serum proteins in an anion-exchange-based purification of Cohn supernatant I. Consequently, the presented process-data-based PLS model calibration in combination with the tool for selective inline quantification has a great potential for the monitoring of future chromatography steps and may contribute to manage batch-to-batch variability by real-time pooling decisions.

  18. 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.

  19. Research Advances on the Process and Application of Whey Protein%乳清蛋白及其加工利用的研究进展

    Institute of Scientific and Technical Information of China (English)



    乳清蛋白作为优质的蛋白质来源,以其良好的功能特性受到了人们的广泛关注,其生产形式多样、应用范围广泛.本文主要综述了乳清蛋白的几种加工方法及其应用现状.%More attention was paid for the major properties of whey protein as a source of protein which was high quality and vaired forms of exploitation with a wide rang of application.This article reviewed several processing methods and the application of whey protein.

  20. A role for Lte1p (a low temperature essential protein involved in mitosis) in proprotein processing in the yeast secretory pathway. (United States)

    Zhao, Xiang; Chang, Amy Y; Toh-E, Akio; Arvan, Peter


    We previously identified six single gene disruptions in Saccharomyces cerevisiae that allow enhanced immunoreactive insulin secretion primarily because of defective Kex2p-mediated endoproteolytic processing. Five eis mutants disrupted established VPS (vacuolar protein sorting) genes, The sixth, LTE1, is a Low Temperature (temperature mitotic defect of lte1. By sequence analysis, Tem1p has highest similarity to Vps21p (yeast homolog of mammalian Rab5). Unlike TEM1, LTE1 is not restricted to mitosis but is expressed throughout the cell cycle. Lte1p function in interphase cells is largely unknown. Here we confirm the eis phenotype of lte1 mutant cells and demonstrate a defect in proalpha factor processing that is rescued by expression of full-length Lte1p but not a C-terminally truncated Lte1p lacking its GEF homology domain. Neither overexpression of Tem1p nor 13 other structurally related GTPases can suppress the secretory proprotein processing defect. However, overexpression of Vps21p selectively restores proprotein processing in a manner dependent upon the active GTP-bound form of the GTPase. By contrast, a vps21 mutant produces a synthetic defect with lte1 in proprotein processing, as well as a synthetic growth defect. Together, the data underscore a link between the mitotic regulator, Lte1p, and protein processing and trafficking in the secretory/endosomal system.

  1. Quantification of the main digestive processes in ruminants: the equations involved in the renewed energy and protein feed evaluation systems. (United States)

    Sauvant, D; Nozière, P


    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.

  2. [Physical arrangement of membrane lipids susceptible to being used in the process of cell sorting of proteins]. (United States)

    Wolf, C; Quinn, P; Koumanov, K; Chachaty, C; Tenchov, B


    Detection of immiscible lipid domains in biological membranes offers an alternative support to protein sorting. Liquid ordered domains ("rafts") comprising cholesterol and saturated sphingolipids incorporate saturated glycosyl-phosphatidylinositol (GPI)-anchored or acylated (palmitoyl- and myristoyl-) proteins or particular transmembrane protein sequences. These lipid domains can be isolated in the form of Detergent resistant membranes (DRM) from biological plasma membrane preparations. Caveolae appear to be a differentiated fraction of plasma membranes comprising such numerous cross-linked microdomains associated with caveolin in different cell types. While the biological relevance of such membrane domains is evidenced in vivo by co-patching of proteins sharing the identical affinity for sphingolipids and by the disruption of co-patching following cell cholesterol depletion, only a few physical studies confort the principle of membrane heterogeneity. Results are now presented where cholesterol addition in a tertiary lipid mixture forces outphase-separation, as a realistic model where the lipid segregation can promote protein sorting to the segregated Lo phase. A lipid mixture comprising phosphatidylserine, phosphatidylethanolamine and sphingomyelin of natural origin in the ratio (1/4/3: mole/mole) has been rendered neatly heterogeneous after the addition of cholesterol (27 mole%). Xray diffraction (Small angle Xray scattering) showed the splitting of two neatly resolved lamellar diffractions in the presence of cholesterol. Above 37 degrees C the heterogeneity was traceable by a broadened diffraction spot up to the complete get-to-liquid transition of sphingomyelin at temperatures > 40 degrees C where the spot became again symmetrical and narrow. The large temperature range where the immiscible lamellar phases are detected, the specific requirement for cholesterol association with sphingomyelin, the positive influence of calcium and the reversibility of domain

  3. 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. (United States)

    Yu, Gloria Qingyu; Warkentin, Tom; Niu, Zhiyuan; Khan, Nazir A; Yu, Peiqiang


    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.

  4. 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. (United States)

    Alam, Syed Imteyaz; Dixit, Aparna; Tomar, Arvind; Singh, Lokendra


    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.

  5. Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent. (United States)

    Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen


    Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc.

  6. Targeting Vascular Endothelial Growth Factor Receptor 2 and Protein Kinase D1 Related Pathways by a Multiple Kinase Inhibitor in Angiogenesis and Inflammation Related Processes In Vitro


    Attila Varga; Pál Gyulavári; Zoltán Greff; Krisztina Futosi; Tamás Németh; Laura Simon-Szabó; Krisztina Kerekes; Csaba Szántai-Kis; Diána Brauswetter; Márton Kokas; Gábor Borbély; Anna Erdei; Attila Mócsai; György Kéri; Tibor Vántus


    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific...

  7. Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Gullberg, Maria; Li, Jiong;


    The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3Cpro) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3Cpro can be expected to be produced at equivalent concentrations. However, using...... production of diagnostic reagents and improved vaccines against foot-and-mouth disease....

  8. Processing of the 5'-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA. (United States)

    Kuroda, Hiroshi; Sugiura, Masahiro


    The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b 6 /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5'-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5'-UTRs, the psbN 5'-UTR has two processing sites, at -39 and -24 upstream from the initiation site. Processing at -39 enhanced the translation rate fivefold. In contrast, processing at -24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5'-UTR has no Shine-Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5'-UTR or with deleted psbN 5'-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5'-UTR. Mobility shift assays using 10 other chloroplast 5'-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.

  9. Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification. (United States)

    Zheng, Ji; Wang, Lu; Twarowska, Barbara; Laino, Sarah; Sparks, Colleen; Smith, Timothy; Russell, Reb; Wang, Michelle


    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.

  10. Behavior of Escherichia coli bacteria in whey protein and corn meal during twin screw extrusion processing at different temperatures (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 ...

  11. Enrichment and purification of casein glycomacropeptide from whey protein isolate using supercritical carbon dioxide processing and membrane filtration (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. 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


    Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen-capturing cell towards a professional antigen presenting cells. In this study, a 2-D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expressi...

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

    DEFF Research Database (Denmark)

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


    repair (HRR), their precise role(s) within this pathway remains poorly understood. Here, we have identified a specific role for the Shu proteins in a Rad51/Rad54-dependent HRR pathway(s) to repair MMS-induced lesions during S-phase. We show that, although mutation of RAD51 or RAD54 prevented...

  14. Amino acid profile of raw and locally processed seeds of Prosopis africana and Ricinus communis: potential antidotes to protein malnutrition

    Directory of Open Access Journals (Sweden)

    Chidi U. Igwe


    Full Text Available Background: Increasing incidence of malnutrition occasioned by high incidence of hunger,worsening food situation in the world, insufficient availability and high cost of animal protein sources, has necessitated extensive research into and use of alternative plant protein sources especially underexploited leguminous seeds.Methods: Flours from raw, boiled and fermented seeds of Prosopis africana and Ricinus communis were evaluated for crude protein and amino acid (AA profiles, and their protein qualities determined. Results: Fermentation improved the protein contents of raw seeds of P. africana and R. communis by 18.70% and 3.95% respectively. In the raw and fermented P. africana seeds, glutamate at 132.60 ± 1.30 and 182.70 ± 3.02 mg/g crude protein (mg/gcp was the most abundant amino acid (AA, while leucine (62.80 ± 0.60 and 79.50 ± 2.01 mg/gcp was the most concentrated essential amino acid (EAA. Aspartate (151.90 ± 2.01 and 170.10 ± 2.00 mg/gcp and arginine (72.80 ± 2.01 and 78.60 ± 2.00 mg/gcp were the most concentrated and abundant non-essential amino acid (NEAA and EAA in the raw and fermented samples of R. communisrespectively. The total AA concentrations (mg/gcp of raw and fermented P. africana were 733.00 and 962.60 respectively, while those of R. communis were 823.50 and 894.10 respectively. The total EAA contents (mg/gcp for P. africana were 311.00 (raw and 404.50 (fermented, and for R. communis; 401.10 (raw and 430.30 (fermented. Threonine was the limiting EAA in raw and fermented P. africana, whereas lysine was the limiting EAA in R. communis raw sample. Fermentation significantly (p<0.05 increased the individual AA compositions of P. africana and R. communis by 94% and 53% respectively, while boiling reduced these parameters significantly (p<0.05 by 47% and 82% respectively. Conclusion: P. africana and R. communis seeds are potentially important plant sources of protein and essential amino acids, and so could be of great

  15. PEG-salt aqueous two-phase systems: an attractive and versatile liquid-liquid extraction technology for the downstream processing of proteins and enzymes. (United States)

    Glyk, Anna; Scheper, Thomas; Beutel, Sascha


    Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)-salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid-liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG-salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid-liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.

  16. Development of Electronic Nose and Near Infrared Spectroscopy Analysis Techniques to Monitor the Critical Time in SSF Process of Feed Protein

    Directory of Open Access Journals (Sweden)

    Hui Jiang


    Full Text Available In order to assure the consistency of the final product quality, a fast and effective process monitoring is a growing need in solid state fermentation (SSF industry. This work investigated the potential of non-invasive techniques combined with the chemometrics method, to monitor time-related changes that occur during SSF process of feed protein. Four fermentation trials conducted were monitored by an electronic nose device and a near infrared spectroscopy (NIRS spectrometer. Firstly, principal component analysis (PCA and independent component analysis (ICA were respectively applied to the feature extraction and information fusion. Then, the BP_AdaBoost algorithm was used to develop the fused model for monitoring of the critical time in SSF process of feed protein. Experimental results showed that the identified results of the fusion model are much better than those of the single technique model both in the training and validation sets, and the complexity of the fusion model was also less than that of the single technique model. The overall results demonstrate that it has a high potential in online monitoring of the critical moment in SSF process by use of integrating electronic nose and NIRS techniques, and data fusion from multi-technique could significantly improve the monitoring performance of SSF process.

  17. Development of electronic nose and near infrared spectroscopy analysis techniques to monitor the critical time in SSF process of feed protein. (United States)

    Jiang, Hui; Chen, Quansheng


    In order to assure the consistency of the final product quality, a fast and effective process monitoring is a growing need in solid state fermentation (SSF) industry. This work investigated the potential of non-invasive techniques combined with the chemometrics method, to monitor time-related changes that occur during SSF process of feed protein. Four fermentation trials conducted were monitored by an electronic nose device and a near infrared spectroscopy (NIRS) spectrometer. Firstly, principal component analysis (PCA) and independent component analysis (ICA) were respectively applied to the feature extraction and information fusion. Then, the BP_AdaBoost algorithm was used to develop the fused model for monitoring of the critical time in SSF process of feed protein. Experimental results showed that the identified results of the fusion model are much better than those of the single technique model both in the training and validation sets, and the complexity of the fusion model was also less than that of the single technique model. The overall results demonstrate that it has a high potential in online monitoring of the critical moment in SSF process by use of integrating electronic nose and NIRS techniques, and data fusion from multi-technique could significantly improve the monitoring performance of SSF process.

  18. Application of the quality by design approach to the drug substance manufacturing process of an Fc fusion protein: towards a global multi-step design space. (United States)

    Eon-duval, Alex; Valax, Pascal; Solacroup, Thomas; Broly, Hervé; Gleixner, Ralf; Strat, Claire L E; Sutter, James


    The article describes how Quality by Design principles can be applied to the drug substance manufacturing process of an Fc fusion protein. First, the quality attributes of the product were evaluated for their potential impact on safety and efficacy using risk management tools. Similarly, process parameters that have a potential impact on critical quality attributes (CQAs) were also identified through a risk assessment. Critical process parameters were then evaluated for their impact on CQAs, individually and in interaction with each other, using multivariate design of experiment techniques during the process characterisation phase. The global multi-step Design Space, defining operational limits for the entire drug substance manufacturing process so as to ensure that the drug substance quality targets are met, was devised using predictive statistical models developed during the characterisation study. The validity of the global multi-step Design Space was then confirmed by performing the entire process, from cell bank thawing to final drug substance, at its limits during the robustness study: the quality of the final drug substance produced under different conditions was verified against predefined targets. An adaptive strategy was devised whereby the Design Space can be adjusted to the quality of the input material to ensure reliable drug substance quality. Finally, all the data obtained during the process described above, together with data generated during additional validation studies as well as manufacturing data, were used to define the control strategy for the drug substance manufacturing process using a risk assessment methodology.

  19. EFEK KOLAGEN DARI BERBAGAI JENIS TULANG IKAN TERHADAP KUALITAS MIOFIBRIL PROTEIN IKAN SELAMA PROSES DEHIDRASI [Effect of Various Fish Bone Collagens on the Quality of Myofibril Fish Protein During Dehydration Process

    Directory of Open Access Journals (Sweden)

    Yudhomenggolo Sastro Darmanto*


    Full Text Available Increase in fish fillet export in Indonesia has caused an increase in its waste such as bones, spines, skin and entrails of fish. Fish bones can be processed by demineralization to produce collagen, an important food additive. The effect of addition of 5% of collagen obtained from fresh water, brackish water and sea water fish bone on the fish protein miofibril of grouper was investigated in this research. Water sorption isotherm, Ca-ATPase activity, gel strength, water holding capacity, folding test and viscosity during dehydration process were evaluated. The results showed that collagens made from various fish bones have different Ca-ATPase activity. The reduction rate of Ca-ATPase activity were in accordance with the reduction of water sorbtion isotherm, gel forming ability, water holding capacity, viscosity and folding test during dehydration process.

  20. Effect of increasing the protein-to-fat ratio and reducing fat content on the chemical and physical properties of processed cheese product. (United States)

    Guinee, T P; O'Callaghan, D J


    Scientific studies indicate that the intake of dietary fat and saturated fats in the modern Western diet is excessive and contributes adversely to health, lifestyle, and longevity. In response, manufacturers of cheese and processed cheese products (PCP) are pursuing the development of products with reduced fat contents. The present study investigated the effect of altering the fat level (13.8, 18.2, 22.7, 27.9, and 32.5 g/100g) in PCP on their chemical and physical properties. The PCP were formulated in triplicate to different fat levels using Cheddar cheese, skim milk cheese, anhydrous milk fat, emulsifying salt (ES), NaCl, and water. The formulations were designed to give fixed moisture (~53 g/100g) and ES:protein ratio (0.105). The resultant PCP, and their water-soluble extracts (WSE), prepared from a macerated blend of PCP and water at a weight ratio of 1:2, were analyzed at 4d. Reducing the fat content significantly increased the firmness of the unheated PCP and reduced the flowability and maximum loss tangent (fluidity) of the melted PCP. These changes coincided with increases in the levels of total protein, water-soluble protein, water-insoluble protein, and water-soluble Ca, and a decrease in the molar ratio of water-soluble Ca to soluble P. However, both water-soluble Ca and water-soluble protein decreased when expressed as percentages of total protein and total Ca, respectively, in the PCP. The high level of protein was a major factor contributing to the deterioration in physical properties as the fat content of PCP was reduced. Diluting the protein content or reducing the potential of the protein to aggregate, and thereby form structures that contribute to rigidity, may provide a means for improving quality of reduced-fat PCP by using natural cheese with lower intact casein content and lower calcium:casein ratio, for example, or by decreasing the ratio of sodium phosphate to sodium citrate-based ES.

  1. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process (United States)

    Iacovache, Ioan; de Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoît


    Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.


    Directory of Open Access Journals (Sweden)

    Into Suhardjo


    Full Text Available The progressiveness of malignant tumors influenced by various complex factors. One of the important factors is Tenascin-C (Tn-C protein, which can interact with fibrinectin as an anti adhesive or anti modulation protein. Tenascin-C is an extra cellular matrix glycoprotein (EMG, which can be found in the oral tissue also as an up regulator. They can be associated with EMG, and strongly influenced promotion of the stromal cell as cell growth, migration, differentiation, angiogenesis, and apoptosis in cancer. Alternative splicing of fibronectin-like type III (FN III repeats of Tn-C generates a number of splice variants, and influences tumor progressiveness. The conclusion of Tn-C role in tumor progressiveness depends on the molecular weight and alternative splicing of FN III.

  3. Protein improvement in Gari by the use of pure cultures of microorganisms involved in the natural fermentation process. (United States)

    Ahaotu, I; Ogueke, C C; Owuamanam, C I; Ahaotu, N N; Nwosu, J N


    The ability of microorganisms involved in cassava mash fermentation to produce and improve protein value by these microorganisms during fermentation was studied. Standard microbiological procedures were used to isolate, identify and determine the numbers of the organisms. Alcaligenes faecalis, Lactobacillus plantarum, Bacillus subtilis, Leuconostoc cremoris, Aspergillus niger, A. tamari, Geotrichum candidum and Penicillium expansum were isolated and identified from cassava waste water while standard analytical methods were used to determine the ability of the isolates to produce linamarase and the proximate composition, pH and titrable acidity of the fermenting mash. The linamarase activity of the isolates ranged from 0.0416 to 0.2618 micromol mL(-1) nmol(-1). Bacillus subtilis, A. niger, A. tamari and P. expansum did not express any activity for the enzyme. Protein content of mash fermented with mixed fungal culture had the highest protein value (15.4 mg/g/dry matter) while the raw cassava had the least value (2.37 mg/g/dry matter). The naturally fermented sample had the least value for the fermented samples (3.2 mg/g/dry matter). Carbohydrate and fat contents of naturally fermented sample were higher than values obtained from the other fermented samples. Microbial numbers of the sample fermented with mixed bacterial culture was highest and got to their peak at 48 h (57 x 10(8) cfu g(-1)). pH decreased with increase in fermentation time with the mash fermented by the mixed culture of fungi having the lowest pH of 4.05 at the end of fermentation. Titrable acidity increased with increase in fermentation time with the highest value of 1.32% at 96 h of fermentation produced by the mixed culture of fungi. Thus fermentation with the pure cultures significantly increased the protein content of mash.

  4. 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


    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.

  5. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes**

    Institute of Scientific and Technical Information of China (English)

    Farfán-García Eunice Dalet; Soriano-Ursúa Marvin Antonio


    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that al osteric binding sites are involved in the affinity and selec-tivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifical y, new possibilities are explored in relation to al osteric and orthosteric binding sites on dopamine receptors for the treatment of Parkinson’s disease, and on muscarinic receptors for Alzheimer’s disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopa-mine receptor holds promise as a relevant therapeutic strategy for Parkinson’s disease. Regarding the treatment of Alzheimer’s disease, the design of dualsteric ligands for mono-oligomeric musca-rinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway.

  6. Characterization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate Bayesian approach. (United States)

    Fu, Zhibiao; Baker, Daniel; Cheng, Aili; Leighton, Julie; Appelbaum, Edward; Aon, Juan


    The principle of quality by design (QbD) has been widely applied to biopharmaceutical manufacturing processes. Process characterization is an essential step to implement the QbD concept to establish the design space and to define the proven acceptable ranges (PAR) for critical process parameters (CPPs). In this study, we present characterization of a Saccharomyces cerevisiae fermentation process using risk assessment analysis, statistical design of experiments (DoE), and the multivariate Bayesian predictive approach. The critical quality attributes (CQAs) and CPPs were identified with a risk assessment. The statistical model for each attribute was established using the results from the DoE study with consideration given to interactions between CPPs. Both the conventional overlapping contour plot and the multivariate Bayesian predictive approaches were used to establish the region of process operating conditions where all attributes met their specifications simultaneously. The quantitative Bayesian predictive approach was chosen to define the PARs for the CPPs, which apply to the manufacturing control strategy. Experience from the 10,000 L manufacturing scale process validation, including 64 continued process verification batches, indicates that the CPPs remain under a state of control and within the established PARs. The end product quality attributes were within their drug substance specifications. The probability generated with the Bayesian approach was also used as a tool to assess CPP deviations. This approach can be extended to develop other production process characterization and quantify a reliable operating region. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:799-812, 2016.

  7. Single Molecule Analysis of the Arabidopsis FRA1 Kinesin Shows that It Is a Functional Motor Protein with Unusually High Processivity

    Institute of Scientific and Technical Information of China (English)

    Chuanmei Zhu; Ram Dixit


    The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism.The cortical localization of this kinesin during interphase raises the possibility that it transports cell wallrelated cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning.To determine whether FRA1 is an authentic motor protein,we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant,GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)-GFP).We found that FRA1(707)-GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated by the presence of microtubules.Using single molecule studies,we found that FRA1(707)-GFP moves processively along microtubule tracks at a velocity of about 0.4 μm s-1.In addition,we found that FRA1(707)-GFP is a microtubule plus-end-directed motor and that it moves along microtubules as a dimer.Interestingly,our single molecule analysis shows that the processivity of FRA1(707)-GFP is at least twice the processivity of conventional kinesin,making FRA1 the most processive kinesin to date.Together,our data show that FRA1 is a bona fide motor protein that has the potential to drive long-distance transport of cargo along cortical microtubules.

  8. Size exclusion and reversed-phase high-performance liquid chromatography/UV for routine control of thermal processing of cows' and donkey milk major proteins. (United States)

    Pinho, Carina; Martins, Zita E; Petisca, Catarina; Figurska, Agata M; Pinho, Olívia; Ferreira, Isabel M P L V O


    Cows' and donkey milks (raw and thermally processed) and respective whey were analysed for quantification of major proteins. Two different chromatographic approaches, size exclusion (SE-HPLC) and reversed-phase high performance liquid chromatography (RP-HPLC) both coupled to UV detection were used. Usefulness of these methods for routine control of the effect of thermal processing was evaluated. The external standard method was used to calibrate the SE-HPLC and RP-HPLC systems. Concerning quantification of β-lactoglobulin (β-lg), α-lactalbumin (α-la), lysozyme (lys), and total casein (cn), no significant differences between results obtained by SE-HPLC and by RP-HPLC (t-test, P>0·05) were observed for raw milks and whey. Heating of cows' milk promoted aggregation of denatured proteins as observed by SE-HPLC, whereas α-la and β-lg from donkey milk were stable to thermal processing at 100 °C (5 min). Lys was quantified in donkey raw milk and whey however, in thermally processed donkey milk lys was denatured and could not be quantified by HPLC.

  9. Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling. (United States)

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


    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.

  10. MUC16/CA125 in the Context of Modular Proteins with an Annotated Role in Adhesion-Related Processes: In Silico Analysis

    Directory of Open Access Journals (Sweden)

    Ninoslav Mitic


    Full Text Available Mucin 16 (MUC16 is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is no detailed insight into their heterogeneity and ligand properties, which may greatly influence its function and biomarker potential. This study was aimed at obtaining further insight into the biological capacity of MUC16/CA125, using in silico analysis of corresponding mucin sequences, including similarity searches as well as GO (gene ontology-based function prediction. The results obtained pointed to the similarities within extracellular serine/threonine rich regions of MUC16 to sequences of proteins expressed in evolutionary distant taxa, all having in common an annotated role in adhesion-related processes. Specifically, a homology to conserved domains from the family of herpesvirus major outer envelope protein (BLLF1 was found. In addition, the possible involvement of MUC16/CA125 in carbohydrate-binding interactions or cellular transport of protein/ion was suggested.

  11. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes. (United States)

    Senderskiy, Igor V; Timofeev, Sergey A; Seliverstova, Elena V; Pavlova, Olga A; Dolgikh, Viacheslav V


    Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

  12. Secretion of Antonospora (Paranosema locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

    Directory of Open Access Journals (Sweden)

    Igor V Senderskiy

    Full Text Available Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species, strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

  13. 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.

  14. Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins

    DEFF Research Database (Denmark)

    Sasaki, T; Göhring, W; Mann, K;


    Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4...... in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo....

  15. Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Norén, Ove


    The post-translational processing of pig small-intestinal aminopeptidase N (EC was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo...... for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment...

  16. 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)


    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.

  17. 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


    . 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.......Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3...

  18. Optimization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate Bayesian approach. (United States)

    Fu, Zhibiao; Leighton, Julie; Cheng, Aili; Appelbaum, Edward; Aon, Juan C


    Various approaches have been applied to optimize biological product fermentation processes and define design space. In this article, we present a stepwise approach to optimize a Saccharomyces cerevisiae fermentation process through risk assessment analysis, statistical design of experiments (DoE), and multivariate Bayesian predictive approach. The critical process parameters (CPPs) were first identified through a risk assessment. The response surface for each attribute was modeled using the results from the DoE study with consideration given to interactions between CPPs. A multivariate Bayesian predictive approach was then used to identify the region of process operating conditions where all attributes met their specifications simultaneously. The model prediction was verified by twelve consistency runs where all batches achieved broth titer more than 1.53 g/L of broth and quality attributes within the expected ranges. The calculated probability was used to define the reliable operating region. To our knowledge, this is the first case study to implement the multivariate Bayesian predictive approach to the process optimization for the industrial application and its corresponding verification at two different production scales. This approach can be extended to other fermentation process optimizations and reliable operating region quantitation.

  19. 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


    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 microg TGFBIp per mg wet tissue. Two-dimensional (2-D...... of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced...

  20. Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments. (United States)

    Johnson, A L; Braidotti, P; Pietra, G G; Russo, S J; Kabore, A; Wang, W J; Beers, M F


    Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular

  1. 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


    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.

  2. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex. (United States)

    Spear, Allyn; Ogram, Sushma A; Morasco, B Joan; Smerage, Lucia Eisner; Flanegan, James B


    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showed that 3D entered the replication complex in the form of its precursor, P3 (or 3CD), and was cleaved to release active 3D polymerase. Furthermore, our results showed that P3 is the preferred precursor that binds to the 5'CL. Using reciprocal complementation assays, we showed that one molecule of P3 binds the 5'CL and that a second molecule of P3 provides 3D. In addition, we showed that a second molecule of P3 served as the VPg provider. These results support a model in which P3 binds to the 5'CL and recruits additional molecules of P3, which are cleaved to release either 3D or VPg to initiate RNA replication.

  3. Toxicity, activation process, and histopathological effect of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 on Tuta absoluta. (United States)

    Sellami, Sameh; Cherif, Maroua; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jamoussi, Kaïs


    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 < 0.05). Action mode examination showed that Vip3Aa16 (88 kDa) was more sensitive to proteolysis activation by the chymotrypsin than the trypsin or the 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.

  4. Production and characterization of poly(3-hydroxybutyrate) generated by Alcaligenes latus using lactose and whey after acid protein precipitation process. (United States)

    Berwig, Karina Hammel; Baldasso, Camila; Dettmer, Aline


    Whey after acid protein precipitation was used as substrate for PHB production in orbital shaker using Alcaligenes latus. Statistical analysis determined the most appropriate hydroxide for pH neutralization of whey after protein precipitation among NH4OH, KOH and NaOH 10%w/v. The results were compared to those of commercial lactose. A scale-up test in a 4L bioreactor was done at 35°C, 750rpm, 7L/min air flow, and 6.5 pH. The PHB was characterized through Fourier Transform Infrared Spectroscopy, thermogravimetry and differential scanning calorimetry. NH4OH provided the best results for productivity (p), 0.11g/L.h, and for polymer yield, (YP/S), 1.08g/g. The bioreactor experiment resulted in lower p and YP/S. PHB showed maximum degradation temperature (291°C), melting temperature (169°C), and chemical properties similar to those of standard PHB. The use of whey as a substrate for PHB production did not affect significantly the final product quality.

  5. Prediction of individual milk proteins including free amino acids in bovine milk using mid-infrared spectroscopy and their correlations with milk processing characteristics. (United States)

    McDermott, A; Visentin, G; De Marchi, M; Berry, D P; Fenelon, M A; O'Connor, P M; Kenny, O A; McParland, S


    -infrared spectroscopy predictions (0.95). Weaker correlations among FAA were observed than the correlations among the protein fractions. Pearson correlations between gold standard protein fractions and the milk processing characteristics of rennet coagulation time, curd firming time, curd firmness, heat coagulating time, pH, and casein micelle size were weak to moderate and ranged from -0.48 (protein and pH) to 0.50 (total casein and a30). Pearson correlations between gold standard FAA and these milk processing characteristics were also weak to moderate and ranged from -0.60 (Val and pH) to 0.49 (Val and K20). Results from this study indicate that mid-infrared spectroscopy has the potential to predict protein fractions and some FAA in milk at a population level.

  6. Protein folding, protein homeostasis, and cancer

    Institute of Scientific and Technical Information of China (English)

    John H. Van Drie


    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

  7. Effect of various domestic processing methods on antinutrients and in vitro protein and starch digestibility of two indigenous varieties of Indian tribal pulse, Mucuna pruriens Var. utilis. (United States)

    Siddhuraju, P; Becker, K


    The effect of various domestic processing methods on antinutrients and starch fractions and in vitro protein and starch digestibilities of white and black varieties of Mucuna pruriens var. utilis was studied. Cooking or autoclaving of both raw seeds and presoaked seeds in different solutions (water, tamarind extract, sodium bicarbonate, and citric acid) significantly (p < 0.05) reduced the content of total phenolics, phytic acid, trypsin inhibitor and chymotrypsin inhibitor activities, and L-dopa compared to soaking or dry heating techniques. The germination processes (24 and 48 h) were also effective in the reduction of various antinutrients, although this reduction appeared to be more pronounced in a prolonged period of germination (72 h). Water soaking followed by dehusking was found to be ineffective in the reduction of trypsin and chymotrypsin inhibitor activities in both varieties. All of the treatments were effective in significantly (p < 0.05) reducing the resistant starch content in the presently investigated samples. Cooking as well as autoclaving brought about a more significant (p < 0.05) improvement in the digestibility of protein and starch compared to germination and dry heat treatment. Moreover, among the different processing techniques, soaking in sodium bicarbonate solution followed by cooking (29.6-34.8%) or autoclaving (33.0-37.2%) seemed to be the best method for improving starch digestibility.

  8. The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease. (United States)

    Prasad, Hari; Rao, Rajini


    Early intervention may be key to safe and effective therapies in patients with Alzheimer disease. Endosomal dysfunction is an early step in neurodegeneration. Endosomes are a major site of production of Aβ peptide from the processing of amyloid precursor protein (APP) by clipping enzymes (β- and γ-secretases). The β-secretase enzyme BACE1 requires acidic lumen pH for optimum function, and acid pH promotes Aβ aggregation. The Na(+)/H(+) exchanger NHE6 provides a leak pathway for protons, limiting luminal acidification by proton pumps. Like APP, NHE6 expression was induced upon differentiation of SH-SY5Y neuroblastoma cells and localized to an endosomal compartment. Therefore, we investigated whether NHE6 expression altered APP localization and processing in a stably transfected cell culture model of human APP expression. We show that co-expression with NHE6 or treatment with the Na(+)/H(+) ionophore monensin shifted APP away from the trans-Golgi network into early and recycling endosomes in HEK293 cells. NHE6 alkalinized the endosomal lumen, similar to monensin, and significantly attenuated APP processing and Aβ secretion. In contrast, Aβ production was elevated upon NHE6 knockdown. We show that NHE6 transcript and protein levels are lowered in Alzheimer brains relative to control. These findings, taken together with emerging genetic evidence linking endosomal Na(+)/H(+) exchangers with Alzheimer disease, suggest that proton leak pathways may regulate Aβ generation and contribute to disease etiology.

  9. 螺旋藻色素蛋白复合物的分离工艺%Separation process of Spirulina pigment-protein complex

    Institute of Scientific and Technical Information of China (English)

    闫岩; 傅红; 杨琳; 张新明


    The separation process of spirulina pigment-protein complex was studied.Through ultrasonic fragmentation,the crude spirulina pigment-protein complex(SPP) was extracted.The experiment compared the effect of ammonium sulfate precipitation and isoelectric point precipitation by measuring the concentration of soluble protein and phycocyanin.Different processes were compared by UV-vis spectrum scan and SDS-PAGE,and phycocyanin with a purity of 0.89 considering as a food grade was obtained by adjusting pH value for impurities precipitation.The result showed that the method had high application value to be commercial process in food industry to obtain phycocyanin from spirulina.%对螺旋藻色素蛋白的分离工艺进行了研究。使用超声波破碎螺旋藻获得螺旋藻色素蛋白复合物粗提液。以可溶性蛋白和藻蓝蛋白浓度为指标,研究硫酸铵沉降和等点点沉降2种分离方法对蛋白分离的影响。通过光谱扫描和SDS-PAGE比较,使用调节pH沉淀杂蛋白分离方法,能得到藻蓝蛋白纯度达到0.89的食品级色素蛋白复合物。结果表明,此方法适合规模化分离螺旋藻中的藻蓝蛋白,在食品工业中有一定的应用前景。

  10. Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase. (United States)

    Sticher, L; Hofsteenge, J; Neuhaus, J M; Boller, T; Meins, F


    The fungicidal class I chitinases (EC are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

  11. Penicillium antifungal protein (PAF) is involved in the apoptotic and autophagic processes of the producer Penicillium chrysogenum. (United States)

    Kovács, Barbara; Hegedűs, Nikoletta; Bálint, Mihály; Szabó, Zsuzsa; Emri, Tamás; Kiss, Gréta; Antal, Miklós; Pócsi, István; Leiter, Eva


    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.

  12. A novel family of katanin-like 2 protein isoforms (KATNAL2), interacting with nucleotide-binding proteins Nubp1 and Nubp2, are key regulators of different MT-based processes in mammalian cells. (United States)

    Ververis, Antonis; Christodoulou, Andri; Christoforou, Maria; Kamilari, Christina; Lederer, Carsten W; Santama, Niovi


    Katanins are microtubule (MT)-severing AAA proteins with high phylogenetic conservation throughout the eukaryotes. They have been functionally implicated in processes requiring MT remodeling, such as spindle assembly in mitosis and meiosis, assembly/disassembly of flagella and cilia and neuronal morphogenesis. Here, we uncover a novel family of katanin-like 2 proteins (KATNAL2) in mouse, consisting of five alternatively spliced isoforms encoded by the Katnal2 genomic locus. We further demonstrate that in vivo these isoforms are able to interact with themselves, with each other and moreover directly and independently with MRP/MinD-type P-loop NTPases Nubp1 and Nubp2, which are integral components of centrioles, negative regulators of ciliogenesis and implicated in centriole duplication in mammalian cells. We find KATNAL2 localized on interphase MTs, centrioles, mitotic spindle, midbody and the axoneme and basal body of sensory cilia in cultured murine cells. shRNAi of Katnal2 results in inefficient cytokinesis and severe phenotypes of enlarged cells and nuclei, increased numbers of centrioles and the manifestation of aberrant multipolar mitotic spindles, mitotic defects, chromosome bridges, multinuclearity, increased MT acetylation and an altered cell cycle pattern. Silencing or stable overexpression of KATNAL2 isoforms drastically reduces ciliogenesis. In conclusion, KATNAL2s are multitasking enzymes involved in the same cell type in critically important processes affecting cytokinesis, MT dynamics, and ciliogenesis and are also implicated in cell cycle progression.

  13. 9 CFR 95.4 - Restrictions on the importation of processed animal protein, offal, tankage, fat, glands, certain... (United States)


    ..., and placental liquids derived from ruminants that have been in any region listed in § 94.18(a) of this...) Glands, unprocessed fat tissue, and blood and blood products derived from ruminants; (iii) Processed fats... derived from ruminants. (2) Any of the materials listed in paragraphs (a)(2)(i) through (a)(2)(iv) of...

  14. 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)


    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.

  15. Kiwellin, a modular protein from green and gold kiwi fruits: evidence of in vivo and in vitro processing and IgE binding. (United States)

    Tuppo, Lisa; Giangrieco, Ivana; Palazzo, Paola; Bernardi, Maria Livia; Scala, Enrico; Carratore, Vito; Tamburrini, Maurizio; Mari, Adriano; Ciardiello, M Antonietta


    Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

  16. The Effect of Oral Intake of Low-Temperature-Processed Whey Protein Concentrate on Colitis and Gene Expression Profiles in Mice

    Directory of Open Access Journals (Sweden)

    Sharmila Jayatilake


    Full Text Available Inflammatory bowel disease (IBD is an autoimmune disease of unknown etiology and can lead to inflammation and cancer. Whey proteins contain many bioactive peptides with potential health benefits against IBD. We investigated the effect of low-temperature-processed whey protein concentrate (LWPC on the suppression of IBD by using a dextran sodium sulfate (DSS-induced colitis model in BALB/c mice. Oral intake of LWPC resulted in improved recovery of body weight in mice. Histological analysis showed that the epithelium cells of LWPC-treated mice were healthier and that lymphocyte infiltration was reduced. The increase in mucin due to the LWPC also reflected reduced inflammation in the colon. Transcriptome analysis of the colon by DNA microarrays revealed marked downregulation of genes related to immune responses in LWPC-fed mice. In particular, the expression of interferon gamma receptor 2 (Ifngr2 and guanylate-binding proteins (GBPs was increased by DSS treatment and decreased in LWPC-fed mice. These findings suggest that LWPCs suppress DSS-induced inflammation in the colon by suppressing the signaling of these cytokines. Our findings suggest that LWPCs would be an effective food resource for suppressing IBD symptoms.

  17. 酶法提取玉米胚芽蛋白的工艺优化%Corn germ protein enzymatic extraction process optimization

    Institute of Scientific and Technical Information of China (English)

    皮钰珍; 刘彦妮; 岳喜庆; 尹伊


    玉米胚芽蛋白是一种优质植物蛋白,其必需氨基酸种类齐全,营养价值高,同时具有多种生理功能,在食品加工中可作为一种理想的植物蛋白质添加物,研究了使用酶法提取玉米胚芽蛋白的工艺参数,通过单因素实验和响应曲面法确定了提取玉米胚芽蛋白的最佳工艺条件:料液比1:6.8g/mL,加酶量3000u/g料,酶解温度60℃,酶解时间180min,pH7.0,此条件玉米胚芽蛋白的提取率可达到31.06%。%Corn germ protein is a high quality vegetable protein,essential amino acid full range of its high nutritional value,but also has a variety of physiological functions;in food processing plants can be used as an ideal protein supplement.In this paper,the us

  18. Effects of various feed supplements containing fish protein hydrolysate or fish processing by-products on the innate immune functions of juvenile coho salmon (oncorhynchus kisutch) (United States)

    Murray, A.L.; Pascho, R.J.; Alcorn, S.W.; Fairgrieve, W.T.; Shearer, K.D.; Roley, D.


    Immunomodulators administered to fish in the diet have been shown in some cases to enhance innate immune defense mechanisms. Recent studies have suggested that polypeptide fractions found in fish protein hydrolysates may stimulate factors in fish important for disease resistance. For the current study, groups of coho salmon were reared on practical feeds that contained either fish meal (Control diet), fish meal supplemented with cooked fish by-products, or fish meal supplemented with hydrolyzed fish protein alone, or with hydrolyzed fish protein and processed fish bones. For each diet group, three replicate tanks of fish were fed the experimental diets for 6 weeks. Morphometric measurements, and serologic and cellular assays were used to evaluate the general health and immunocompetence of fish in the various feed groups. Whereas the experimental diets had no effect on the morphometric and cellular measurements, fish fed cooked by-products had increased leucocrit levels and lower hematocrit levels than fish from the other feed groups. Innate cellular responses were increased in all feed groups after feeding the four experimental diets compared with pre-feed results. Subgroups of fish from each diet group were also challenged with Vibrio anguillarum (ca. 7.71 ?? 105 bacteria ml-1) at 15??C by immersion. No differences were found in survival among the various feed groups.

  19. Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate. (United States)

    Curti, Elena; Seid, Christopher A; Hudspeth, Elissa; Center, Lori; Rezende, Wanderson; Pollet, Jeroen; Kwityn, Cliff; Hammond, Molly; Matsunami, Rise K; Engler, David A; Hotez, Peter J; Elena Bottazzi, Maria


    Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation

  20. 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


    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.

  1. Quick generation of Raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture. (United States)

    Berry, Brandon N; Dobrowsky, Terrence M; Timson, Rebecca C; Kshirsagar, Rashmi; Ryll, Thomas; Wiltberger, Kelly


    Mitigating risks to biotherapeutic protein production processes and products has driven the development of targeted process analytical technology (PAT); however implementing PAT during development without significantly increasing program timelines can be difficult. The development of a monoclonal antibody expressed in a Chinese hamster ovary (CHO) cell line via fed-batch processing presented an opportunity to demonstrate capabilities of altering percent glycated protein product. Glycation is caused by pseudo-first order, non-enzymatic reaction of a reducing sugar with an amino group. Glucose is the highest concentration reducing sugar in the chemically defined media (CDM), thus a strategy controlling glucose in the production bioreactor was developed utilizing Raman spectroscopy for feedback control. Raman regions for glucose were determined by spiking studies in water and CDM. Calibration spectra were collected during 8 bench scale batches designed to capture a wide glucose concentration space. Finally, a PLS model capable of translating Raman spectra to glucose concentration was built using the calibration spectra and spiking study regions. Bolus feeding in mammalian cell culture results in wide glucose concentration ranges. Here we describe the development of process automation enabling glucose setpoint control. Glucose-free nutrient feed was fed daily, however glucose stock solution was fed as needed according to online Raman measurements. Two feedback control conditions were executed where glucose was controlled at constant low concentration or decreased stepwise throughout. Glycation was reduced from ∼9% to 4% using a low target concentration but was not reduced in the stepwise condition as compared to the historical bolus glucose feeding regimen.

  2. Laboratory Experiment Optimization of Desalting and De-protein Process for Enteromorpha Polysaccharides%浒苔多糖脱蛋白脱盐方法的优选

    Institute of Scientific and Technical Information of China (English)

    陈达妙; 林文庭