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Sample records for extracted proteins accumulate

  1. Protein Extractability

    African Journals Online (AJOL)

    human nutrition, particularly in developing ... studied the effect of NaCl on the extractability of ... significant influence on the functional properties ..... Malaka, S. L. O. and Amund, O. O. Studies on the Life Cycle and Morphometrics of Honeybees,.

  2. Differential Protein Accumulation in Banana Fruit during Ripening 1

    Science.gov (United States)

    Dominguez-Puigjaner, Eva; Vendrell, Miguel; Ludevid, M. Dolors

    1992-01-01

    Banana (Musa acuminata, cv Dwarf Cavendish) proteins were extracted from pulp tissue at different stages of ripening and analyzed by two-dimensional electrophoresis. The results provide evidence of differential protein accumulation during ripening. Two sets of polypeptides have been detected that increase substantially in ripe fruit. These polypeptides were characterized as glycoproteins by western blotting and concanavalin A binding assays. Antibodies againts tomato polygalacturonase cross-react with one of these sets of proteins. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668607

  3. Oil and Protein Accumulation in Soybean Grains under Salinity Stress

    Directory of Open Access Journals (Sweden)

    Kazem GHASSEMI-GOLEZANI

    2010-06-01

    Full Text Available Two factorial experiments based on randomized complete block design (RCBD with three replications were conducted in 2007 and 2008, to evaluate grain development (four harvests and rate and duration of oil and protein accumulation in three soybean cultivars (�Williams�, �Zan� and �L17� under a non-saline (control and three saline (3, 6 and 9 ds/m NaCl conditions. Six seeds were sown in each pot filled with 900 g perlite, using 144 pots for each experiment. After emergence, seedlings were thinned and 4 plants were kept in each pot. Rate of oil accumulation up to maturity was not significantly affected by salinity. With increasing salinity, rate and percentage of protein accumulation, duration of oil and protein accumulation and oil and protein content per grain decreased, but oil percentage increased. Oil and protein yields per plant decreased as salinity increased. These reductions were mainly attributed to the short duration of protein and oil accumulation and grain yield per plant under saline conditions. �Williams� had the highest rate and duration of protein accumulation and rate of oil accumulation, but �L17� had the highest grain yield per plant. Consequently, differences in protein and oil yields per plant between these two cultivars were not statistically significant. However, �Zan� had the lowest protein and oil yields, due to the lowest grain yield per plant.

  4. Accumulation of Pathogenesis-related Type-5 Like Proteins in Phytoplasma infected Garland Chrysanthemum Chrysanthemum coronarium

    Institute of Scientific and Technical Information of China (English)

    Bo-Xiong ZHONG; Yan-Wei SHEN

    2004-01-01

    Soluble proteins extracted from leaves, apical shoots, axillary shoots, and stems of garland chrysanthemum plants infected by onion yellows phytoplasma were analyzed by two-dimensional gel electrophoresis. Computerized matching analysis revealed that at least six soluble proteins were accumulated specifically in phytoplasma-infected garland chrysanthemum. N-terminal amino acids sequences of these soluble proteins, determined by Edman degradation, shared high sequence similarities with those ofpathogenesis-related type-5 (PR-5) proteins such as tobacco thaumatin-like protein. Accumulation of these six proteins was also found in garland chrysanthemum plants infected by other phytoplasmas. These results demonstrate that phytoplasmal infection induces the accumulation of PR-5 like proteins in garland chrysanthemum plants.

  5. Relationship between Protein Accumulation and Nitrogen Accumulation and Translocation in Different Genotype Soybean

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Physiological studies of soybean [Glycine max(L.)Merr.]genotypes with wide differences in seed protein concentration may permit detection of important yield-related processes.In order to research the law of protein accumulation and the characteristics of N accumulation and translocation,we did an experiment with three soybean cultivars which have different protein content and the similar phase in pot culture.The results showed that the laws of protein accumulation of three soybean cultivars are similar in the course of seeding;protein content descended in the early stage,and increased steadily in the middle period,then increased quickly in the later period.But the speed of protein accumulation in soybean seeds was difference in different period.In addition,high-protein cultivar (Dongnong 42) and intermediate protein cultivar (Dongnong 7819) were more than those of low-protein cultivar (Dongnong 434),including nitrogen contents in leaf and petiole,stem and pod,peak value of nitrogen accumulation of the whole plant,value of nitrogen translocation,its efficiency.

  6. Metabolic adaptation in transplastomic plants massively accumulating recombinant proteins.

    Directory of Open Access Journals (Sweden)

    Julia Bally

    Full Text Available BACKGROUND: Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Here we used proteomics to characterize tobacco (Nicotiana tabacum plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD or a green fluorescent protein (GFP. While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO(2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. CONCLUSIONS/SIGNIFICANCE: The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation.

  7. Automatic Extraction of Protein Interaction in Literature

    OpenAIRE

    Liu, Peilei; Wang, Ting

    2014-01-01

    Protein-protein interaction extraction is the key precondition of the construction of protein knowledge network, and it is very important for the research in the biomedicine. This paper extracted directional protein-protein interaction from the biological text, using the SVM-based method. Experiments were evaluated on the LLL05 corpus with good results. The results show that dependency features are import for the protein-protein interaction extraction and features related to the interaction w...

  8. Accumulation of small protein molecules in a macroscopic complex coacervate

    NARCIS (Netherlands)

    Lindhoud, S.; Claessens, M.M.A.E.

    2016-01-01

    To obtain insight into the accumulation of proteins into macroscopic complex coacervate phases, the lysozyme concentration in complex coacervates containing the cationic polyelectrolyte poly-(N,N dimethylaminoethyl methacrylate) and the anionic polyelectrolyte polyacrylic acid was investigated as a

  9. Motor protein accumulation on antiparallel microtubule overlaps

    CERN Document Server

    Kuan, Hui-Shun

    2015-01-01

    Biopolymers serve as one-dimensional tracks on which motor proteins move to perform their biological roles. Motor protein phenomena have inspired theoretical models of one-dimensional transport, crowding, and jamming. Experiments studying the motion of Xklp1 motors on reconstituted antiparallel microtubule overlaps demonstrated that motors recruited to the overlap walk toward the plus end of individual microtubules and frequently switch between filaments. We study a model of this system that couples the totally asymmetric simple exclusion process (TASEP) for motor motion with switches between antiparallel filaments and binding kinetics. We determine steady-state motor density profiles for fixed-length overlaps using exact and approximate solutions of the continuum differential equations and compare to kinetic Monte Carlo simulations. The center region, far from the overlap ends, has a constant motor density as one would na\\"ively expect. However, rather than following a simple binding equilibrium, the center ...

  10. [Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

    Science.gov (United States)

    Błachnio, Karina

    2010-01-01

    Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

  11. How biomass composition determines protein extractability

    NARCIS (Netherlands)

    Sari, Y.W.; Syafitri, U.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    Biomass consists of a complex mixture of different components, of which protein potentially has a high added value for biorefinery. In this study, protein extractability of different types of biomass, mostly by-products, was analyzed. Protein yield obtained from a three step extraction using alkali

  12. How biomass composition determines protein extractability

    NARCIS (Netherlands)

    Sari, Y.W.; Syafitri, U.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    Biomass consists of a complex mixture of different components, of which protein potentially has a high added value for biorefinery. In this study, protein extractability of different types of biomass, mostly by-products, was analyzed. Protein yield obtained from a three step extraction using alkali

  13. Plasmodesmal Targeting and Accumulation of TMV Movement Protein.

    Science.gov (United States)

    Wright, Kathryn M; Chapman, Sean; Roberts, Alison G

    2007-05-01

    The interaction between tobacco mosaic virus and its host plant cells has been intensively studied as a model for macromolecular trafficking. The observation that GFP-labelled TMV movement protein localises to microtubules led to the suggestion that microtubules are required for the cell to cell movement of the virus. In a recent paper we have demonstrated that the targeting of TMV movement protein to plasmodesmata requires the actin and ER networks, which supports previous evidence from our laboratory that showed that disruption of microtubules did not prevent cell to cell movement of TMV virus, and that a mutated movement protein, which did not localise to micro-tubules, showed enhanced viral movement. In this addendum we speculate where the TMV movement protein accumulates within plasmodesmata, and the relationship of this accumulation to the cell to cell movement of the virus.

  14. Cloning of matrix Gla protein in a marine cartilaginous fish, Prionace glauca: preferential protein accumulation in skeletal and vascular systems.

    Science.gov (United States)

    Ortiz-Delgado, J B; Simes, D C; Viegas, C S B; Schaff, B J; Sarasquete, C; Cancela, M L

    2006-07-01

    Matrix Gla protein (MGP) belongs to the family of vitamin K dependent, Gla containing proteins and, in mammals, birds and Xenopus, its mRNA has been previously detected in bone, cartilage and soft tissue extracts, while the accumulation of the protein was found mainly in calcified tissues. More recently, the MGP gene expression was also studied in marine teleost fish where it was found to be associated with chondrocytes, smooth muscle and endothelial cells. To date no information is available on the sites of MGP expression or accumulation in cartilaginous fishes that diverged from osteichthyans, a group that includes mammals, over 400 million years ago. The main objectives of this work were to study the sites of MGP gene expression and protein accumulation by means of in situ hybridization and immunohistochemistry. MGP mRNA and protein were localized as expected not only in cartilage from branchial arches and vertebra but also in the endothelia of the vascular system as well as in the tubular renal endothelium. The accumulation of MGP in non mineralized soft tissues was unexpected and suggests differences in localization or regulation of this protein in shark soft tissues compared to tetrapods and teleosts. Our results also corroborate the hypothesis that in Prionace glauca, as previously shown in mammals, the MGP protein probably also acts as a calcification inhibitor, protecting soft tissues from abnormal and ectopic calcification.

  15. Relationship between Protein Accumulation Regulation and Yield Formation in Soybean

    Institute of Scientific and Technical Information of China (English)

    CHEN Lihua; LI Jie; LIU Lijun; ZU Wei

    2006-01-01

    Three different genotypes soybeans were adopted in this experiment under three fertilizer levels.The object of this study was to investigate protein accumulation regulation of soybean cultivars under the condition of different nutrient levels, and their effects on soybean yield and quality, and to provide theoretical evidence for breed, cultivation and agricultural production, also man-powered controllable locations. The concentration of N in the leaves declined after seedling stage, then increased again at stage of early flowering, and started to decrease up to leaf senescence, declined rapidly from seed-filling season to stage of yellow ripeness. The concentration of N in the stems and pod walls declined with growth stage. High seed protein genotypes exhibited higher N assimilating and partitioning during whole growth stages. Pod walls were media of N partitioning. Protein was accumulated mainly during the later period of reproductive growth stage up to harvest, so plant growth after stage of yellow ripeness could not be neglected.

  16. Optimization of Protein Extraction for Lichen Thalli.

    Science.gov (United States)

    Kondratiuk, Anna S; Savchuk, Oleksiy M; Hur, Jae-Seoun

    2015-06-01

    Lichen-forming fungal proteins have been seldom searched due to many difficulties in their extraction. Phenols, quinones, proteases, and other components released during cell disruption have been known to be the greatest challenges related to protein extraction from lichens. To overcome these problems and maintain good electrophoretic resolution and high protein concentration, an extraction buffer containing polyvinylpolypyrrolidone, ascorbic acid, Triton X-100, polyethylene glycol, proteinase, and oxidase inhibitors in sodium phosphate buffer was developed. This extraction buffer showed high efficiency for all lichen species tested in the study.

  17. TDP-43 regulates endogenous retrovirus-K viral protein accumulation.

    Science.gov (United States)

    Manghera, Mamneet; Ferguson-Parry, Jennifer; Douville, Renée N

    2016-10-01

    The concomitant expression of neuronal TAR DNA binding protein 43 (TDP-43) and human endogenous retrovirus-K (ERVK) is a hallmark of ALS. Since the involvement of TDP-43 in retrovirus replication remains controversial, we sought to evaluate whether TDP-43 exerts an effect on ERVK expression. In this study, TDP-43 bound the ERVK promoter in the context of inflammation or proteasome inhibition, with no effect on ERVK transcription. However, over-expression of ALS-associated aggregating forms of TDP-43, but not wild-type TDP-43, significantly enhanced ERVK viral protein accumulation. Human astrocytes and neurons further demonstrated cell-type specific differences in their ability to express and clear ERVK proteins during inflammation and proteasome inhibition. Astrocytes, but not neurons, were able to clear excess ERVK proteins through stress granule formation and autophagy. In vitro findings were validated in autopsy motor cortex tissue from patients with ALS and neuro-normal controls. We further confirmed marked enhancement of ERVK in cortical neurons of patients with ALS. Despite evidence of enhanced stress granule and autophagic response in ALS cortical neurons, these cells failed to clear excess ERVK protein accumulation. This highlights how multiple cellular pathways, in conjunction with disease-associated mutations, can converge to modulate the expression and clearance of viral gene products from genomic elements such as ERVK. In ALS, ERVK protein aggregation is a novel aspect of TDP-43 misregulation contributing towards the pathology of this neurodegenerative disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Effect of Ultrasound in Soybean Protein Extraction

    Science.gov (United States)

    Fukase, Hirokazu; Ohdaira, Etsuzo; Masuzawa, Nobuyoshi; Ide, Masao

    1994-05-01

    Application of ultrasound for accelerating the extraction of nutriments in food processing has been attempted. However, conditions of exposure to ultrasound were not clear in previous studies. This paper reports on the relationship between the ultrasonic pressure and the amount of extracted protein from soybeans. Experiments were conducted using a beaker, in which the ultrasonic fields were precisely measured. Soybean flakes suspended in water were put in the beaker and placed in a water tank. The amount of extracted protein in water upon ultrasonic exposure was calculated by the Kjeldahl method. It was found that the amount of extracted protein increased in proportion to ultrasonic pressure up to the total amount of soybean protein soluble in water. Furthermore, this paper describes the denaturation of the protein produced by the ultrasonic cavitation.

  19. Research of lead extract method from accumulator scrap

    Directory of Open Access Journals (Sweden)

    Віктор Юрійович Кушнеров

    2016-12-01

    Full Text Available The analysis of production and consumption of lead from primary and secondary raw material is executed. It is determined that approximately 56 % general world lead production consists of lead-content secondary raw material processing – scrap and wastes of lead and accumulator scrap. Using thermodynamic analysis, it is determined that potassium bicarbonate has an advantage at processing of sulfate constituent of secondary raw material

  20. Screening for bioactive metabolites in plant extracts modulating glucose uptake and fat accumulation

    DEFF Research Database (Denmark)

    El-Houri, Rime Bahij; Kotowska, Dorota Ewa; C. B. Olsen, Louise

    2014-01-01

    while weekly activating PPARγ without promoting adipocyte differentiation. In addition, these extracts were able to decrease fat accumulation in C. elegans. Methanol extracts of summer savory (Satureja hortensis), common elder, and broccoli (Brassica oleracea) enhanced glucose uptake in myotubes...... but were not able to activate PPARγ, indicating a PPARγ-independent effect on glucose uptake....

  1. Sentence Simplification Aids Protein-Protein Interaction Extraction

    CERN Document Server

    Jonnalagadda, Siddhartha

    2010-01-01

    Accurate systems for extracting Protein-Protein Interactions (PPIs) automatically from biomedical articles can help accelerate biomedical research. Biomedical Informatics researchers are collaborating to provide metaservices and advance the state-of-art in PPI extraction. One problem often neglected by current Natural Language Processing systems is the characteristic complexity of the sentences in biomedical literature. In this paper, we report on the impact that automatic simplification of sentences has on the performance of a state-of-art PPI extraction system, showing a substantial improvement in recall (8%) when the sentence simplification method is applied, without significant impact to precision.

  2. A Novel Approach for Protein-Named Entity Recognition and Protein-Protein Interaction Extraction

    OpenAIRE

    Meijing Li; Tsendsuren Munkhdalai; Xiuming Yu; Keun Ho Ryu

    2015-01-01

    Many researchers focus on developing protein-named entity recognition (Protein-NER) or PPI extraction systems. However, the studies about these two topics cannot be merged well; then existing PPI extraction systems’ Protein-NER still needs to improve. In this paper, we developed the protein-protein interaction extraction system named PPIMiner based on Support Vector Machine (SVM) and parsing tree. PPIMiner consists of three main models: natural language processing (NLP) model, Protein-NER mod...

  3. Olive Leaf Extract from Sicilian Cultivar Reduced Lipid Accumulation by Inducing Thermogenic Pathway during Adipogenesis

    Science.gov (United States)

    Palmeri, Rosa; Monteleone, Julieta I.; Spagna, Giovanni; Restuccia, Cristina; Raffaele, Marco; Vanella, Luca; Li Volti, Giovanni; Barbagallo, Ignazio

    2016-01-01

    Olive leaves contain a wide variety of phenolic compounds belonging to phenolic acids, phenolic alcohols, flavonoids, and secoiridoids, and include also many other pharmacological active compounds. They could play an important role in human diet and health because of their ability to lower blood pressure, increase coronary arteries blood flow and decrease the risk of cardiovascular diseases. The aim of this study was to investigate the effect of olive leaf extract (OLE) from Sicilian cultivar on adipogenic differentiation of human adipose derived mesenchymal stem cells and its impact on lipid metabolism. We showed that OLE treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein uncoupling protein 1, sirtuin 1, peroxisome proliferator-activated receptor alpha, and coactivator 1 alpha. Furthermore, OLE significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. Taken together, our results suggest that OLE may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis. PMID:27303302

  4. Accumulation of some metals by legumes and their extractability from acid mine spoils. [USA - Alabama

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, R.W.; Ibeabuchi, I.O.; Sistani, K.R.; Shuford, J.W. (Alabama A M University, Normal, AL (USA). Dept. of Plant and Soil Science)

    A greenhouse study was conducted to investigate the growth (dry matter yield) of selected legume cover crops; phytoaccumulation of metals such as Zn, Mn, Pb, Cu, Ni, and Al; the extractability of heavy metals from three different Alabama acid mine spoils. The spoils were amended based on soil test recommended levels of N, P, K, Ca and Mg prior to plant growth. Metals were extracted by three extractants (Mehlich 1, DTPA, and 0.1 M HCl) and values correlated with their accumulation by the selected legumes. Among the cover crops, kobe lespedeza {ital Lespedeza striata} (Thung.) Hook and Arn, sericea lespedeza {ital Lespedeza cuneata} (Dum.) G. Don, and red clover (Trifolium pratense L.) did not survive the stressful conditions of the spoils. However, cowpea (Vigna unguiculata L.) followed by Bragg' soybean {ital Glycine max} (L.) Merr. generally produced the highest dry matter yield while accumulating the largest quantity of metals, except Al, from spoils. The extractability of most metals from the spoils was generally in the order of: 0.1 MHCl {gt} DTPA. Mehlich 1 did not extract Pb and 0.1 M HCl did not extract Ni, whereas DTPA extracted all the metals in a small amount relative to HCl and Mehlich 1. All the extractants were quite effective in removing plant-available Zn from the spoils. In general, the extractants' ability to predict plant-available metals depended on the crop species, spoil type, and extractant used. 28 refs., 4 tabs.

  5. Yam (Dioscorea batatas) Root and Bark Extracts Stimulate Osteoblast Mineralization by Increasing Ca and P Accumulation and Alkaline Phosphatase Activity.

    Science.gov (United States)

    Kim, Suji; Shin, Mee-Young; Son, Kun-Ho; Sohn, Ho-Yong; Lim, Jae-Hwan; Lee, Jong-Hwa; Kwun, In-Sook

    2014-09-01

    Yam (Dioscorea batatas) is widely consumed as functional food for health promotion mainly in East Asia countries. We assessed whether yam root (tuber) or bark (peel) extracts stimulated the activity of osteoblasts for osteogenesis. MC3T3-E1 cells (mouse osteoblasts) were treated with yam root extracts (water or methanol) (study I) or bark extracts (water or hexane) (study II) within 0~10 μg/mL during the periods of osteoblast proliferation (5~10 day), matrix maturation (11~15 day) and mineralization (16~20 day) as appropriate. In study I, both yam root water and methanol extracts increased cell proliferation as concentration-dependent manner. Cellular collagen synthesis and alkaline phosphatase (ALP) activity, both the indicators of bone matrix protein and inorganic phosphate production for calcification respectively, were also increased by yam root water and methanol extract. Osteoblast calcification as cell matrix Ca and P accumulation was also increased by the addition of yam root extracts. In study II, yam bark extracts (water and hexane) increased osteoblast proliferation and differentiation, as collagen synthesis and ALP activity and osteoblast matrix Ca and P deposition. The study results suggested that both yam root and bark extracts stimulate osteogenic function in osteoblasts by stimulating bone matrix maturation by increasing collagen synthesis, ALP activity, and matrix mineralization.

  6. Soluble Moringa oleifera leaf extract reduces intracellular cadmium accumulation and oxidative stress in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kerdsomboon, Kittikhun; Tatip, Supinda; Kosasih, Sattawat; Auesukaree, Choowong

    2016-05-01

    Moringa oleifera leaves are a well-known source of antioxidants and traditionally used for medicinal applications. In the present study, the protective action of soluble M. oleifera leaf extract (MOLE) against cadmium toxicity was investigated in the model eukaryote Saccharomyces cerevisiae. The results showed that this extract exhibited a protective effect against oxidative stress induced by cadmium and H2O2 through the reduction of intracellular reactive oxygen species. Interestingly, not only the co-exposure of soluble MOLE with cadmium but also pretreatment of this extract prior to cadmium exposure significantly reduced the cadmium uptake through an inhibition of Fet4p, a low-affinity iron(II) transporter. In addition, the supplementation of soluble MOLE significantly reduced intracellular iron accumulation in a Fet4p-independent manner. Our findings suggest the potential use of soluble extract from M. oleifera leaves as a dietary supplement for protection against cadmium accumulation and oxidative stress.

  7. Extracting knowledge from protein structure geometry

    DEFF Research Database (Denmark)

    Røgen, Peter; Koehl, Patrice

    2013-01-01

    to minima of their free energy surfaces. It is well known however that the situation is more complicated as the current force fields used for molecular simulations fail to recognize native states from misfolded structures. In an attempt to solve this problem, we follow an alternate approach and derive a new...... potential from geometric knowledge extracted from native and misfolded conformers of protein structures. This new potential, Metric Protein Potential (MPP), has two main features that are key to its success. Firstly, it is composite in that it includes local and nonlocal geometric information on proteins...

  8. Absence of upregulated genes associated with protein accumulations in desmin myopathy.

    Science.gov (United States)

    Raju, Raghavan; Dalakas, Marinos C

    2007-03-01

    In desmin myopathy but not hereditary inclusion-body myopathy (hIBM), there is accumulation of myofibrillar proteins including desmin, myotilin, dystrophin, gelsolin, actin, and CDC kinase. To assess the cause of protein excess, we studied the genes coding the accumulated proteins in desmin myopathy, hIBM, and controls. No differences were found among them. In desmin myopathy, protein accumulation is not due to upregulation of genes triggered by mutant desmin, but rather to posttranslational disassembly of intermediate filaments.

  9. Prion protein accumulation in lipid rafts of mouse aging brain.

    Directory of Open Access Journals (Sweden)

    Federica Agostini

    Full Text Available The cellular form of the prion protein (PrP(C is a normal constituent of neuronal cell membranes. The protein misfolding causes rare neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases. These maladies can be sporadic, genetic or infectious. Sporadic prion diseases are the most common form mainly affecting aging people. In this work, we investigate the biochemical environment in which sporadic prion diseases may develop, focusing our attention on the cell membrane of neurons in the aging brain. It is well established that with aging the ratio between the most abundant lipid components of rafts undergoes a major change: while cholesterol decreases, sphingomyelin content rises. Our results indicate that the aging process modifies the compartmentalization of PrP(C. In old mice, this change favors PrP(C accumulation in detergent-resistant membranes, particularly in hippocampi. To confirm the relationship between lipid content changes and PrP(C translocation into detergent-resistant membranes (DRMs, we looked at PrP(C compartmentalization in hippocampi from acid sphingomyelinase (ASM knockout (KO mice and synaptosomes enriched in sphingomyelin. In the presence of high sphingomyelin content, we observed a significant increase of PrP(C in DRMS. This process is not due to higher levels of total protein and it could, in turn, favor the onset of sporadic prion diseases during aging as it increases the PrP intermolecular contacts into lipid rafts. We observed that lowering sphingomyelin in scrapie-infected cells by using fumonisin B1 led to a 50% decrease in protease-resistant PrP formation. This may suggest an involvement of PrP lipid environment in prion formation and consequently it may play a role in the onset or development of sporadic forms of prion diseases.

  10. Olive leaf extract from Sicilian cultivar reduced lipid accumulation by inducing thermogenic pathway during adipogenesis

    Directory of Open Access Journals (Sweden)

    Rosa ePalmeri

    2016-05-01

    Full Text Available Olive leaves contain a wide variety of phenolic compounds belonging to phenolic acids, phenolic alcohols, flavonoids and secoiridoids, and include also many other pharmacological active compounds. They could play an important role in human diet and health because of their ability to lower blood pressure, increase coronary arteries blood flow and decrease the risk of cardiovascular diseases. The aim of this study was to investigate the effect of Olive leaf extract (OLE from Sicilian cultivar on adipogenic differentiation of human adipose derived mesenchymal stem cells and its impact on lipid metabolism. We showed that OLE treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein UCP1, SIRT-1, PPARα and Pgc-1α. Furthermore, OLE significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. Taken together, our results suggest that OLE may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis.

  11. Influence of extraction method on protein profile of soybeans

    OpenAIRE

    Pavlićević Milica Ž.; Stanojević Slađana P.; Vucelić-Radović Biljana V.

    2013-01-01

    Comparison between protein profiles of soybean obtained by commonly used methods of extraction (Tris buffer and Tris-urea buffer) with methods used for extraction of plant proteins for 2D PAGE analysis (direct solubilization in IEF buffer, acetone extraction, phenol extraction, extraction with urea solubilization buffer and thiourea-urea extraction) was investigated. 2D profiles of samples extracted directly in IEF buffer, in urea solubilization buffer and in acetone were characterized ...

  12. Reduction of lipid accumulation in white adipose tissues by Cassia tora (Leguminosae) seed extract is associated with AMPK activation.

    Science.gov (United States)

    Tzeng, Thing-Fong; Lu, Hung-Jen; Liou, Shorong-Shii; Chang, Chia Ju; Liu, I-Min

    2013-01-15

    Natural herbal medications may be one answer to the worldwide epidemic of obesity. This study examines the effects of Cassia seed ethanol extract (CSEE) upon lipid accumulation in white adipose tissue (WAT). CSEE exhibited a significant concentration-dependent decrease in the intracellular accumulation of trigycerides in 3T3-L1 adipocytes. After being fed a high-fat diet (HFD) for 2 weeks, rats were fed CSEE (100, 200 or 300 mg/kg) once daily for 8 weeks. CSEE caused dose-related reductions in body weight gain (as well as plasma lipid levels and epididymal WAT sizes in HFD-fed rats). CSEE enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream targeting enzyme, acetyl-CoA carboxylase, up-regulated gene expression of carnitine palmitoyl transferase 1, and down-regulated sterol regulatory element-binding protein 1 and fatty acid synthase protein levels in epididymal WAT of HFD-fed rats. CSEE could attenuate lipid accumulation in WAT via AMPK signaling pathway activation.

  13. comparison of protein extraction methods for the leaves of ficus ...

    African Journals Online (AJOL)

    F. I. Abdullah

    2017-05-01

    May 1, 2017 ... degradation. Based on the results of ... Eze and Dumboff [30] reported that the addition of chlorophyll extract to the standard protein .... protein extraction from recalcitrant plants such as banana (Musa spp.), apple (Malus ...

  14. Rosehip Extract Inhibits Lipid Accumulation in White Adipose Tissue by Suppressing the Expression of Peroxisome Proliferator-activated Receptor Gamma.

    Science.gov (United States)

    Nagatomo, Akifumi; Nishida, Norihisa; Matsuura, Yoichi; Shibata, Nobuhito

    2013-06-01

    Recent studies have shown that Rosa canina L. and tiliroside, the principal constituent of its seeds, exhibit anti-obesity and anti-diabetic activities via enhancement of fatty acid oxidation in the liver and skeletal muscle. However, the effects of rosehip, the fruit of this plant, extract (RHE), or tiliroside on lipid accumulation in adipocytes have not been analyzed. We investigated the effects of RHE and tiliroside on lipid accumulation and protein expression of key transcription factors in both in vitro and in vivo models. RHE and tiliroside inhibited lipid accumulation in a dose-dependent manner in 3T3-L1 cells. We also analyzed the inhibitory effect of RHE on white adipose tissue (WAT) in high-fat diet (HFD)-induced obesity mice model. Male C57BL/6J mice were fed HFD or HFD supplemented with 1% RHE (HFDRH) for 8 weeks. The HFDRH-fed group gained less body weight and had less visceral fat than the HFD-fed group. Liver weight was significantly lower in the HFDRH-fed group and total hepatic lipid and triglyceride (TG) content was also reduced. A significant reduction in the expression of peroxisome proliferator-activated receptor gamma (PPARγ) was observed in epididymal fat in the HFDRH-fed group, in comparison with controls, through Western blotting. These results suggest that downregulation of PPARγ expression is involved, at least in part, in the suppressive effect of RHE on lipid accumulation in WAT.

  15. Soil metaproteomics – Comparative evaluation of protein extraction protocols

    OpenAIRE

    Keiblinger, Katharina M.; Wilhartitz, Inés C.; Schneider, Thomas; Roschitzki, Bernd; Schmid, Emanuel; Eberl, Leo; Riedel, Kathrin; Zechmeister-Boltenstern, Sophie

    2012-01-01

    Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extractio...

  16. Stepwise magma migration and accumulation processes and their effect on extracted melt chemistry

    Directory of Open Access Journals (Sweden)

    Urtson, Kristjan

    2009-12-01

    Full Text Available Numerical and analogue models suggest that melt production, its segregation from the solid matrix and subsequent transport and accumulation are highly dynamic and stepwise processes exhibiting scale invariant patterns in both time and length scales, which is characteristic of self-organized critical systems. This phenomenon is also observed in migmatites at several localities, where the leucosome thickness statistics obey power laws. Stepwise melt transport and deformation-enhanced melt mobility affect melt production dynamics by determining the distribution of extracted melt batch sizes and residence times of melt pockets within the host rock, which in turn would influence the geochemistry of extracted melts. We introduce a numerical approach, which enables qualitative and quantitative assessment of the effects of stress-induced melt migration and accumulation on the chemistry of partial melts. The model suggests that apart from different sources and melting percentages, deformation can be an important factor in producing geochemical variations within and between intrusive/extrusive complexes.

  17. Monitoring Protein and Starch Accumulation in Wheat Grains with Leaf SPAD and Canopy Spectral Reflectance

    Institute of Scientific and Technical Information of China (English)

    TIAN Yong-chao; ZHU yan; CAO Wei-xing; FAN Xue-mei; LIU Xiao-jun

    2003-01-01

    The research was conducted to determine the relationships of protein and starch accumulation dynamics in grains of wheat to post-heading leaf SPAD values and canopy spectral reflectance. The results showed that leaf nitrogen accumulation was exponentially related to leaf SPAD values and linearly related to canopy spectral reflectance, and that there was negative linear relationship between leaf nitrogen accumulation and grain protein accumulation, but positive linear relationship between post-heading leaf nitrogen translocation and grain protein accumulation at maturity. In addition, leaf SPAD values were parabolically related with and ratio indices R(1 500,610) and R(1 220,560) were exponentially related with protein and starch accumulation in grains. These results indicate that leaf SPAD values and canopy spectral reflectance should be good indicators of quality formation dynamics in wheat grains.

  18. Extraction of plant proteins for two-dimensional electrophoresis

    OpenAIRE

    Granier, Fabienne

    1988-01-01

    Three different extraction procedures for two-dimensional electrophoresis of plant proteins are compared: (i) extraction of soluble proteins with a nondenaturing Tris-buffer, (ii) denaturing extraction in presence of sodium dodecyl sulfate at elevated temperature allowing the solubilization of membrane proteins in addition to a recovery of soluble proteins, and (iii) a trichloroacetic acid-acetone procedure allowing the direct precipitation of total proteins.

  19. Accumulation of Protein Fractions during Grain Filloing of Wheat Genotypes Differing in Protein Content and Baking Quality

    Institute of Scientific and Technical Information of China (English)

    LiuXiaobing; LiWenxiong; 等

    1995-01-01

    The accumulation of protein fractions was analyzed on developing and mature wheat grains of three cultivars differing in protein content and baking quality.There was a slight difference in the accumulation of cytoplasmic proteins in the cultivars used.The high yield but low protein cultivar showed a consistent decline of protein content during grain filling but the high-protein cultivars increascd their psotein contant after 25 days post-anthesis.The accumulation of storage proteins was different from that of cytoplasmic protein.and there were also cultivar variations,However,all cultivars reached their.Maximum-synthesizing capacity for storage proteins at maturity.The relationship between the protein fractions or their ratio and baking quality was also discussed.

  20. Linguistic feature analysis for protein interaction extraction

    Directory of Open Access Journals (Sweden)

    Cornelis Chris

    2009-11-01

    Full Text Available Abstract Background The rapid growth of the amount of publicly available reports on biomedical experimental results has recently caused a boost of text mining approaches for protein interaction extraction. Most approaches rely implicitly or explicitly on linguistic, i.e., lexical and syntactic, data extracted from text. However, only few attempts have been made to evaluate the contribution of the different feature types. In this work, we contribute to this evaluation by studying the relative importance of deep syntactic features, i.e., grammatical relations, shallow syntactic features (part-of-speech information and lexical features. For this purpose, we use a recently proposed approach that uses support vector machines with structured kernels. Results Our results reveal that the contribution of the different feature types varies for the different data sets on which the experiments were conducted. The smaller the training corpus compared to the test data, the more important the role of grammatical relations becomes. Moreover, deep syntactic information based classifiers prove to be more robust on heterogeneous texts where no or only limited common vocabulary is shared. Conclusion Our findings suggest that grammatical relations play an important role in the interaction extraction task. Moreover, the net advantage of adding lexical and shallow syntactic features is small related to the number of added features. This implies that efficient classifiers can be built by using only a small fraction of the features that are typically being used in recent approaches.

  1. Protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm of cereals.

    Science.gov (United States)

    Zheng, Yankun; Wang, Zhong

    2014-10-01

    There are mainly three endosperm storage tissues in the cereal endosperm: aleurone cells, sub-aleurone cells and the center starch endosperm. The protein accumulation is very different in the three endosperm storage tissues. The aleurone cells accumulate protein in aleurone granules. The sub-aleurone cells and the center starch endosperm accumulate protein in endoplasmic reticulum-derived protein bodies and vacuolar protein bodies. Proteins are deposited in different patterns within different endosperm storage tissues probably because of the special storage properties of these tissues. There are several special genes and other molecular factors to mediate the protein accumulation in these tissues. Different proteins have distinct functions in the protein body formation and the protein interactions determine protein body assembly. There are both cooperation and competition relationships between protein, starch and lipid in the cereal endosperm. This paper reviews the latest investigations on protein accumulation in aleurone cells, sub-aleurone cells and the center starch endosperm. Useful information will be supplied for future investigations on the cereal endosperm development.

  2. Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95*

    Science.gov (United States)

    Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J.; Alkon, Daniel L.

    2016-01-01

    Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95S295) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. PMID:27330081

  3. Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components

    OpenAIRE

    Patanasethanont, Denpong; NAGAI, Junya; Matsuura, Chie; Fukui, Kyoko; Sutthanut, Khaetthareeya; Sripanidkulchai, Bung-Orn; Yumoto, Ryoko; Takano, Mikihisa

    2007-01-01

    In this study, the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on multidrug resistance associated-proteins (MRP)-mediated transport in A549 cells were examined. The cells employed express MRP1 and MRP2, but not P-glycoprotein. The cellular accumulation of calcein, an MRP substrate, was significantly increased by various MRP inhibitors without being affected by verapamil, a typical P-glycoprotein inhibitor. Ethanol and aqueous extracts from Kaempferia ...

  4. Investigation of relationship between lipid and Monascus pigment accumulation by extractive fermentation.

    Science.gov (United States)

    Wang, Bo; Zhang, Xuehong; Wu, Zhenqiang; Wang, Zhilong

    2015-10-20

    Fermented Monascus pigments have been utilized as traditional Chinese medicine and food colorant for thousands of years. Under the limited nitrogen concentration and/or low initial pH 2.5 conditions, it was observed that production of intracellular pigments and accumulation of microbial lipids (high content reaching to approximately 50% in dry cell weight) by edible Monascus anka exhibited a positive correlated relationship. Extractive fermentation in nonionic surfactant micelle aqueous solution selectively exported the intracellular Monascus pigments into its extracellular broth, in which the concentration of intracellular pigments was negligible while the extracellular one was enhanced. The extractive fermentation provides a novel strategy for shifting of the metabolic channeling from intracellular lipid accumulation to Monascus pigment production. High pigment concentration, i.e., approximately 40 AU of extracellular Monascus pigments, was achieved by extractive fermentation at a relatively high nonionic surfactant concentration 10 g/l. This phenomenon might be attributed to the nonionic surfactant micelles acting as pigment reservoirs by biomimetic of intracellular lipids.

  5. Soil metaproteomics - Comparative evaluation of protein extraction protocols.

    Science.gov (United States)

    Keiblinger, Katharina M; Wilhartitz, Inés C; Schneider, Thomas; Roschitzki, Bernd; Schmid, Emanuel; Eberl, Leo; Riedel, Kathrin; Zechmeister-Boltenstern, Sophie

    2012-11-01

    Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS-phenol extraction and (d) SDS-phenol extraction with prior washing steps. To assess the suitability of these methods for the functional analysis of the soil metaproteome, they were applied to a potting soil high in organic matter and a forest soil. Proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) and the number of unique spectra as well as the number of assigned proteins for each of the respective protocols was compared. In both soil types, extraction with SDS-phenol (c) resulted in "high" numbers of proteins. Moreover, a spiking experiment was conducted to evaluate protein recovery. To this end sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans. The protein recovery in the spiking experiment was almost 50%. Our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils.

  6. Monoclonal antibodies against accumulation-associated protein affect EPS biosynthesis and enhance bacterial accumulation of Staphylococcus epidermidis.

    Directory of Open Access Journals (Sweden)

    Jian Hu

    Full Text Available Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap that contains sequence repeats known as G5 domains, which are responsible for the Zn(2+-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5 were generated. MAb(18B6 inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb(25C11 and MAb(20B9 enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb(18B6, which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb(25C11 and MAb(20B9. Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections.

  7. Selective extraction of isolated mitotic apparatus. Evidence that typical microtubule protein is extracted by organic mercurial.

    Science.gov (United States)

    Bibring, T; Baxandall, J

    1971-02-01

    Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

  8. p53 gene mutations, p53 protein accumulation and compartmentalization in colorectal adenocarcinoma.

    OpenAIRE

    1995-01-01

    p53 accumulation may occur in the nucleus and/or cytoplasm of neoplastic cells. Cytoplasmic accumulation has been reported to be an unfavorable, but not established, prognostic indicator in colorectal cancer. Different types of p53 intracellular compartmentalization could depend either on p53 gene mutations or on the interaction with p53 protein ligands. The purposes of our study were (1) to assess whether the different patterns of p53 accumulation are selectively associated with p53 mutation...

  9. Production of surgical gloves from low extractable protein RVNRL

    Energy Technology Data Exchange (ETDEWEB)

    Marga, Utama; Yanti, S.; Made, Sumarti; Marsongko; Tita, Puspitasari; Dian, Iramani [Center for Research and Development of Isotopes and Radiation Technology, National Nuclear Energy Agency, Jakarta (Indonesia); Makuuchi, K. [EB System Cooperation, Takasaki, Gunma (Japan); Yoshii, F. [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment; Siswanto [Research Unit for Biotechnology of Estate Crop (Indonesia)

    2001-03-01

    Study on the production of surgical gloves from low extractable protein PVNRL (Radiation Vulcanization of Natural Rubber Latex) in home industry scale with normal butyl acrylate as sensitizer has been carried out. The variation of dipping speed, concentration of coagulant agent and selection of antioxidant for producing good quality of surgical gloves were evaluated. The water-extractable protein and PBS (Phosphate Buffer Saline) - extractable protein content, the physical and mechanical properties of gloves were measured. The results show that for producing a good quality of surgical gloves from low extractable protein RVNRL, the concentration of latex is 50% with calcium nitrate as coagulant agent between 15-20%. By using this condition the physical and mechanical properties of surgical gloves is required to ASTM standard such as tensile strength more than 24 MPa, PBS-extractable protein is around 41-68 ug/g and water-extractable protein contents is around 23-35 ug/g. (author)

  10. Integration of galacturonic acid extraction with alkaline protein extraction from green tea leaf residue

    NARCIS (Netherlands)

    Zhang, Chen; Bozileva, Elvira; Klis, van der Frits; Dong, Yiyuan; Sanders, Johan P.M.; Bruins, Marieke E.

    2016-01-01

    Leaf pectin can be used as a feedstock for galacturonic acid (GA) production, but high extraction costs limit economic feasibility. To improve the extraction efficiency, leaf pectin extraction was integrated with an already cost-effective alkaline protein extraction, focusing on high yield of GA

  11. Mitotic apparatus: the selective extraction of protein with mild acid.

    Science.gov (United States)

    Bibring, T; Baxandall, J

    1968-07-26

    The treatment of isolated mitotic apparatus with mild (pH 3) hydrochloric acid results in the extraction of less than 10 percent of its protein, accompanied by the selective morphological disappearance of the microtubules. The same extraction can be shown to dissolve outer doublet microtubules from sperm flagella. A protein with points of similarity to the flagellar microtubule protein is the major component of the extract from mitotic apparatus.

  12. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus

    Directory of Open Access Journals (Sweden)

    Alexandra Martins dos Santos Soares

    Full Text Available Abstract The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract, SE (shell extract and CE (cotyledon extract. Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL–1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL–1. The effective concentration for 50% hatching inhibition (EC50 was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL–1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

  13. Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

    Science.gov (United States)

    Soares, Alexandra Martins dos Santos; de Araújo, Sandra Alves; Lopes, Suzana Gomes; Costa Junior, Livio Martins

    2015-01-01

    The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

  14. Investigation and optimization of the factors influencing sorghum protein extraction.

    Science.gov (United States)

    Park, Seok-Ho; Bean, Scott Roger

    2003-11-19

    To optimize the extraction of sorghum proteins, several variables were examined: sample-to-solvent ratio, detergent type and concentration, reducing agent type and concentration, extraction time, and buffer pH and concentration. Samples were quantified and characterized by RP-HPLC, FZCE, and nitrogen analysis. These studies revealed that pH, detergent type, reducing agent type, and sample-to-solvent ratio all had significant effects on the levels of protein extracted. Increasing SDS concentration (2%) and solvent-to-flour ratio (20:1) with multiple 5 min extracts reduced extraction time by 35-80% while still extracting the same levels of total protein relative to the control methodology. Reproducibility using the multiple extractions was found to be excellent with relative standard deviations of extractions.

  15. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties.

    Science.gov (United States)

    Zhao, Xue; Vázquez-Gutiérrez, José Luis; Johansson, Daniel P; Landberg, Rikard; Langton, Maud

    2016-01-01

    A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE). These results demonstrate that the functional properties of YMPE can be modified for different food applications.

  16. Yellow Mealworm Protein for Food Purposes - Extraction and Functional Properties.

    Directory of Open Access Journals (Sweden)

    Xue Zhao

    Full Text Available A protocol for extraction of yellow mealworm larvae proteins was established, conditions were evaluated and the resulting protein extract was characterised. The freeze-dried yellow mealworm larvae contained around 33% fat, 51% crude protein and 43% true protein on a dry matter basis. The true protein content of the protein extract was about 75%, with an extraction rate of 70% under optimised extraction conditions using 0.25 M NaOH, a NaOH solution:ethanol defatted worm ratio of 15:1 mL/g, 40°C for 1 h and extraction twice. The protein extract was a good source of essential amino acids. The lowest protein solubility in distilled water solution was found between pH 4 and 5, and increased with either increasing or decreasing pH. Lower solubility was observed in 0.5 M NaCl solution compared with distilled water. The rheological tests indicated that temperature, sample concentration, addition of salt and enzyme, incubation time and pH alterations influenced the elastic modulus of yellow mealworm protein extract (YMPE. These results demonstrate that the functional properties of YMPE can be modified for different food applications.

  17. Accumulation of yellow Monascus pigments by extractive fermentation in nonionic surfactant micelle aqueous solution.

    Science.gov (United States)

    Xiong, Xu; Zhang, Xuehong; Wu, Zhenqiang; Wang, Zhilong

    2015-02-01

    Monascus species can produce various secondary metabolites of polyketide structure. In the current study, it is found that an interesting phenomenon, i.e., submerged culture of Monascus species in an aqueous solution majorly accumulated intracellular orange Monascus pigments exhibiting one peak at 470 nm with absorbance of 32 OD while extractive fermentation in a nonionic surfactant micelle aqueous solution produced extracellular and intracellular yellow Monascus pigments exhibiting one peak at 410 nm with absorbance 30 OD and 12 OD, respectively. The spectrum profiles of both intracellular and extracellular Monascus pigments were affected by surfactant loading, extractive fermentation time, and surfactant adding time. Meanwhile, the instability of orange Monascus pigments in the extracellular nonionic surfactant micelle aqueous solution was also confirmed experimentally. The mechanism behind this phenomenon is attributed to the export of intracellular yellow Monascus pigments into its broth by extractive fermentation. The transferring of intracellular yellow Monascus pigments into its broth blocks yellow Monascus pigments from further enzymatic conversion or eliminates the feedback inhibition of yellow Monascus pigments based on the biosynthetic pathway of Monascus pigments.

  18. Benzo(a)pyrene accumulation in soils of technogenic emission zone by subcritical water extraction method

    Science.gov (United States)

    Sushkova, Svetlana; Minkina, Tatiana; Kizilkaya, Ridvan; Mandzhieva, Saglara; Batukaev, Abdulmalik; Bauer, Tatiana; Gulser, Coskun

    2016-04-01

    The purpose of research is the assessment of main marker of polycyclic aromatic hydrocarbons contamination, benzo[a]pyrene (BaP) content in soils of emission zone of the power complex plant in soils with use of ecologically clean and effective subcritical water extraction method. Studies were conducted on the soils of monitoring plots subjected to Novocherkassk Power Plant emissions from burning coal. In 2000, monitoring plots were established at different distances from the NPS (1.0-20.0 km). Soil samples for the determination of soil properties and the contents of BaP were taken from a depth of 0-20 cm. The soil cover in the region under study consisted of ordinary chernozems, meadow-chernozemic soils, and alluvial meadow soils. This soil revealed the following physical and chemical properties: Corg-3.1-5.0%, pH-7.3-7.6, ECE-31.2-47.6 mmol(+)/100g; CaCO3-0.2-1.0%, the content of physical clay - 51-67% and clay - 3-37%. BaP extraction from soils was carried out by a subcritical water extraction method. Subcritical water extraction of BaP from soil samples was conducted in a specially developed extraction cartridge made of stainless steel and equipped with screw-on caps at both ends. It was also equipped with a manometer that included a valve for pressure release to maintain an internal pressure of 100 atm. The extraction cartridge containing a sample and water was placed into an oven connected to a temperature regulator under temperature 250oC and pressure 60 atm. The BaP concentration in the acetonitrile extract was determined by HPLC. The efficiency of BaP extraction from soil was determined using a matrix spike. The main accumulation of pollutant in 20 cm layer of soils is noted directly in affected zone on the plots situated at 1.2, 1.6, 5.0, 8.0 km from emission source in the direction of prevailing winds. The maximum quantity of a pollutant was founded in the soil of the plot located mostly close to a source of pollution in the direction of prevailing winds

  19. Eucommia ulmoides Oliver extract, aucubin, and geniposide enhance lysosomal activity to regulate ER stress and hepatic lipid accumulation.

    Directory of Open Access Journals (Sweden)

    Hwa-Young Lee

    Full Text Available Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia.

  20. Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components.

    Science.gov (United States)

    Patanasethanont, Denpong; Nagai, Junya; Matsuura, Chie; Fukui, Kyoko; Sutthanut, Khaetthareeya; Sripanidkulchai, Bung-orn; Yumoto, Ryoko; Takano, Mikihisa

    2007-07-02

    In this study, the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on multidrug resistance associated-proteins (MRP)-mediated transport in A549 cells were examined. The cells employed express MRP1 and MRP2, but not P-glycoprotein. The cellular accumulation of calcein, an MRP substrate, was significantly increased by various MRP inhibitors without being affected by verapamil, a typical P-glycoprotein inhibitor. Ethanol and aqueous extracts from K. parviflora rhizome increased the accumulation of calcein and doxorubicin in A549 cells in a concentration-dependent manner. The inhibitory potency of the ethanol extract for MRP function was greater than that of the aqueous extract. Among six flavone derivatives isolated from K. parviflora rhizome, 5,7-dimethoxyflavone exhibited a maximal stimulatory effect on the accumulation of doxorubicin in A549 cells. The accumulation of doxorubicin was increased by four flavone derivatives without 5-hydroxy group, but not by the other two flavone derivatives with 5-hydroxy group. In addition, 5,7-dimethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone decreased resistance to doxorubicin in A549 cells. These findings indicate that extracts and flavone derivatives from the rhizome of K. parviflora suppress MRP function, and therefore may be useful as modulators of multidrug resistance in cancer cells.

  1. Comparison of Methods for Protein Extraction from Pine Needles

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Extraction of proteins from pine needles for proteomic analysis has long been a challenge for scientists. We compared three different protein extraction methods including sucrose, Tris-HCl and trichloroacetic acid (TCA)/acetone (TCA method) to determine their efficiency in separating pine needle proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional PAGE (2D-PAGE). Proteins were then separated by SDS-PAGE. Among three methods the method using sucrose extraction buffer showed the highest efficiency and highest quality in separating proteins. In addition, clearer and more stable strips were detected by SDS-PAGE using sucrose extraction buffer. When the proteins extracted using sucrose extraction buffer were separated by 2D-PAGE, more than 300 protein spots, with isoelectric points (PI) ranging from 4.0 to 7.0 and molecular weights (MW) from 6.5 to 97.4 kD, were observed. This confirmed that the method with sucrose extraction buffer was an efficient and reliable method for extracting proteins from pine needles.

  2. Metaproteomics: Evaluation of protein extraction from activated sludge.

    Science.gov (United States)

    Hansen, Susan Hove; Stensballe, Allan; Nielsen, Per Halkjaer; Herbst, Florian-Alexander

    2014-11-01

    Metaproteomic studies of full-scale activated sludge systems require reproducible protein extraction methods. A systematic evaluation of three different extractions protocols, each in combination with three different methods of cell lysis, and a commercial kit were evaluated. Criteria used for comparison of each method included the extracted protein concentration and the number of identified proteins and peptides as well as their phylogenetic, cell localization and functional distribution and quantitative reproducibility. Furthermore, the advantage of using specific metagenomes and a 2-step database approach was illustrated. The results recommend a protocol for protein extraction from activated sludge based on the protein extraction reagent B-Per and bead beating. The data have been deposited to the ProteomeXchange with identifier PXD000862 (http://proteomecentral.proteomexchange.org/dataset/PXD000862).

  3. Preparation of protein samples for gel electrophoresis by sequential extraction

    Institute of Scientific and Technical Information of China (English)

    钟伯雄; 翁宏飚; 等

    2002-01-01

    Since preparation and solubilization of protein samples are crucial factors in proteome research,the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm.Bombyx mori.Two kinds of protein samples were obtained from the body wall using the method.Between the two types of samples only about 15% proteins were identical;the majority were different,indicating that more species of proteins could be obtained with the sequential extraction method;which will be useful for preparation of protein samples for proteome study.

  4. Preparation of protein samples for gel electrophoresis by sequential extraction

    Institute of Scientific and Technical Information of China (English)

    钟伯雄; 翁宏飚; 方维焕

    2002-01-01

    Since preparation and solubilization of protein samples are crucial factors in proteome research, the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm, Bombyx mori. Two kinds of protein samples were obtained from the body wall using the method. Between the two types of samples only about 15% proteins were identical; the majority were different, indicating that more species of proteins could be obtained with the sequential extraction method; which will be useful for preparation of protein samples for proteome study.

  5. Ginkgo biloba extract suppresses hypertrophy and extracellular matrix accumulation in rat mesangial cells

    Institute of Scientific and Technical Information of China (English)

    Jian-yun WANG; Xiao-xing YIN; Yun-ming WU; Dao-quan TANG; Yuan-yuan GAO; Mei-rong WAN; Xiao-yu HOU; Bei ZHANG

    2006-01-01

    Aim: To observe the effects of Ginkgo biloba extract (EGb) on the hypertrophy of mesangial cells and the accumulation of extracellular matrix (ECM) in mesangial cells. Methods: Cultured mesangial cells were allotted into 7 groups: normal group, solvent control group, high glucose group, low dose of EGb group, moderate dose of EGb group, high dose of EGb group, and captopril group. Activities of cell antioxidases, S phase percentage and G0/G1 phase percentage, collagen Ⅳ and laminin, Smad2/3 and Smad7, TGF-β1, Mrna were measured by different methods. Results: For EGb-treated groups, when compared with high glucose group, the cell percentage of S phase was raised and the percentage of G0/G1 was lowered. The intensity of oxidative stress was weakened. The expression of Smad2/3 was greatly decreased and Smad7 was increased. Collagen Ⅳ, laminin and TGF-β1 Mrna were also reduced. Conclusion: EGb can suppress cell hypertrophy and the accumulation of ECM in rat mesangial cells, which means it could play a vital role in the delay of glomerulosclerosis in diabetic nephropathy.

  6. Construction and Power Test of the Extraction Kicker Magnet for the Spallation Neutron Source Accumulator Ring

    CERN Document Server

    Pai, Chien; Hseuh Hsiao Chaun; Meng, Wuzheng; Mi, Jian-Lin; Raparia, Deepak; Sandberg, Jon; Todd, Robert J; Tsoupas, Nicholaos; Tuozzolo, Joseph; Warburton, David S; Wei, Jie; Weiss, Daniel; Yung Lee, Yong; Zhang, Wu

    2005-01-01

    Two extraction kicker magnet assemblies that contain seven individual pulsed magnet modules each will kick the proton beam vertically out of the SNS accumulator ring into the aperture of the extraction lambertson septum magnet. The proton beam then travels to the 1.4 MW SNS target assembly. The 14 kicker magnets and major components of the kicker assembly have been fabricated in BNL. The inner surfaces of the kicker magnets were coated with TiN to reduce the secondary electron yield. All 14 PFN power supplies have been built, tested and delivered to ORNL. Before final installation, a partial assembly of the kicker system with three kicker magnets was assembled to test the functions of each critical component in the system. In this paper we report the progress of the construction of the kicker components, the TiN coating of the magnets, the installation procedure of the magnets and the full power test of the kicker with the PFN power supply.

  7. Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two-dimensional gel electrophoresis.

    Science.gov (United States)

    Parkhey, Suruchi; Chandrakar, Vibhuti; Naithani, S C; Keshavkant, S

    2015-10-01

    Protein extraction for two-dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one-dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77-95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.

  8. Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

    Science.gov (United States)

    Singh, Prashant Kumar; Shrivastava, Alok Kumar; Chatterjee, Antra; Pandey, Sarita; Rai, Snigdha; Singh, Shilpi; Rai, L C

    2015-09-01

    Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

  9. Detergent selection for enhanced extraction of membrane proteins.

    Science.gov (United States)

    Arachea, Buenafe T; Sun, Zhen; Potente, Nina; Malik, Radhika; Isailovic, Dragan; Viola, Ronald E

    2012-11-01

    Generating stable conditions for membrane proteins after extraction from their lipid bilayer environment is essential for subsequent characterization. Detergents are the most widely used means to obtain this stable environment; however, different types of membrane proteins have been found to require detergents with varying properties for optimal extraction efficiency and stability after extraction. The extraction profiles of several detergent types have been examined for membranes isolated from bacteria and yeast, and for a set of recombinant target proteins. The extraction efficiencies of these detergents increase at higher concentrations, and were shown to correlate with their respective CMC values. Two alkyl sugar detergents, octyl-β-d-glucoside (OG) and 5-cyclohexyl-1-pentyl-β-d-maltoside (Cymal-5), and a zwitterionic surfactant, N-decylphosphocholine (Fos-choline-10), were generally effective in the extraction of a broad range of membrane proteins. However, certain detergents were more effective than others in the extraction of specific classes of integral membrane proteins, offering guidelines for initial detergent selection. The differences in extraction efficiencies among this small set of detergents supports the value of detergent screening and optimization to increase the yields of targeted membrane proteins.

  10. Algal Proteins: Extraction, Application, and Challenges Concerning Production

    Directory of Open Access Journals (Sweden)

    Stephen Bleakley

    2017-04-01

    Full Text Available Population growth combined with increasingly limited resources of arable land and fresh water has resulted in a need for alternative protein sources. Macroalgae (seaweed and microalgae are examples of under-exploited “crops”. Algae do not compete with traditional food crops for space and resources. This review details the characteristics of commonly consumed algae, as well as their potential for use as a protein source based on their protein quality, amino acid composition, and digestibility. Protein extraction methods applied to algae to date, including enzymatic hydrolysis, physical processes, and chemical extraction and novel methods such as ultrasound-assisted extraction, pulsed electric field, and microwave-assisted extraction are discussed. Moreover, existing protein enrichment methods used in the dairy industry and the potential of these methods to generate high value ingredients from algae, such as bioactive peptides and functional ingredients are discussed. Applications of algae in human nutrition, animal feed, and aquaculture are examined.

  11. Algal Proteins: Extraction, Application, and Challenges Concerning Production

    Science.gov (United States)

    Bleakley, Stephen; Hayes, Maria

    2017-01-01

    Population growth combined with increasingly limited resources of arable land and fresh water has resulted in a need for alternative protein sources. Macroalgae (seaweed) and microalgae are examples of under-exploited “crops”. Algae do not compete with traditional food crops for space and resources. This review details the characteristics of commonly consumed algae, as well as their potential for use as a protein source based on their protein quality, amino acid composition, and digestibility. Protein extraction methods applied to algae to date, including enzymatic hydrolysis, physical processes, and chemical extraction and novel methods such as ultrasound-assisted extraction, pulsed electric field, and microwave-assisted extraction are discussed. Moreover, existing protein enrichment methods used in the dairy industry and the potential of these methods to generate high value ingredients from algae, such as bioactive peptides and functional ingredients are discussed. Applications of algae in human nutrition, animal feed, and aquaculture are examined. PMID:28445408

  12. Small-scale extraction of recombinant proteins from bacteria.

    Science.gov (United States)

    Simpson, Richard J

    2010-09-01

    Bacteria are particularly convenient for producing recombinant proteins for purification purposes. To monitor induction as well as the levels of recombinant protein expression, it is important to have a rapid, simple method for estimating bacterial protein expression. This protocol describes the preparation of small-scale bacterial extracts using cell lysis with 0.5% Triton X-100.

  13. Protein extraction from xylem and phloem sap.

    Science.gov (United States)

    Kehr, Julia; Rep, Martijn

    2007-01-01

    It is well known that phloem and xylem vessels transport small nutrient molecules over long distances in higher plants. The finding that proteins also occur in both transport fluids was unexpected, and the function of most of these proteins is not yet well understood. This chapter outlines how proteins can be obtained and purified from xylem and phloem saps to perform subsequent proteomic analyses.

  14. Intracellular coagulation inhibits the extraction of proteins from Prochloron

    Science.gov (United States)

    Fall, R.; Lewin, R. A.; Fall, L. R.

    1983-01-01

    Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irreversible loss of cell fragility in the isolated algae. Accompanying this phenomenon, which is termed intracellular coagulation, was a redistribution of thylakoids around the cell periphery, a loss of photosynthetic O2 production, and a drastic decrease in the extractability of cell proteins. Procedures are described for the successful preparation and transport of cell extracts yielding the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as other soluble proteins.

  15. Intracellular coagulation inhibits the extraction of proteins from Prochloron

    Science.gov (United States)

    Fall, R.; Lewin, R. A.; Fall, L. R.

    1983-01-01

    Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irreversible loss of cell fragility in the isolated algae. Accompanying this phenomenon, which is termed intracellular coagulation, was a redistribution of thylakoids around the cell periphery, a loss of photosynthetic O2 production, and a drastic decrease in the extractability of cell proteins. Procedures are described for the successful preparation and transport of cell extracts yielding the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as other soluble proteins.

  16. Fish Muscle Proteins: Extraction, Quantitation, and Electrophoresis

    Science.gov (United States)

    Smith, Denise

    Electrophoresis can be used to separate and visualize proteins. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins are separated based on size. When protein samples are applied to such gels, it is usually necessary to know the protein content of the sample. This makes it possible to apply a volume of sample to the gel such that samples have a comparable amount of total protein. While it is possible to use an official method of protein analysis (e.g., Kjeldahl, N combustion) for such an application, it often is convenient to use a rapid spectroscopic protein analysis that requires only a small amount of sample. The bicinchoninic acid (BCA) assay method will be used for this purpose.

  17. Microbial protein in soil: influence of extraction method and C amendment on extraction and recovery.

    Science.gov (United States)

    Taylor, Erin B; Williams, Mark A

    2010-02-01

    The capacity to study the content and resolve the dynamics of the proteome of diverse microbial communities would help to revolutionize the way microbiologists study the function and activity of microorganisms in soil. To better understand the limitations of a proteomic approach to studying soil microbial communities, we characterized extractable soil microbial proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two methods were utilized to extract proteins from microorganisms residing in a Quitman and Benfield soil: (1) direct extraction of bulk protein from soil and (2) separation of the microorganisms from soil using density gradient centrifugation and subsequent extraction (DGC-EXT) of microbial protein. In addition, glucose and toluene amendments to soil were used to stimulate the growth of a subset of the microbial community. A bacterial culture and bovine serum albumin (BSA) were added to the soil to qualitatively assess their recovery following extraction. Direct extraction and resolution of microbial proteins using SDS-PAGE generally resulted in smeared and unresolved banding patterns on gels. DGC-EXT of microbial protein from soil followed by separation using SDS-PAGE, however, did resolve six to 10 bands in the Benfield but not the Quitman soil. DGC-EXT of microbial protein, but not direct extraction following the addition of glucose and toluene, markedly increased the number of bands (approximately 40) on the gels in both Benfield and Quitman soils. Low recoveries of added culture and BSA proteins using the direct extraction method suggest that proteins either bind to soil organic matter and mineral particles or that partial degradation takes place during extraction. Interestingly, DGC may have been preferentially selected for actively growing cells, as gauged by the 10-100x lower cy19:0/18:1omega7 ratio of the fatty acid methyl esters in the isolated community compared to that for the whole soil. DGC can be used to

  18. Extractable proteins from field radiation vulcanized natural rubber latex

    Energy Technology Data Exchange (ETDEWEB)

    Parra, Duclerc F. [Chemical and Environmental Centre, Nuclear Energy Research Institute, Av. Lineu Prestes, 2242-CEP Sao Paulo (Brazil)]. E-mail: dfparra@ipen.br; Pinto Martins, Carlos Felipe [Chemical and Environmental Centre, Nuclear Energy Research Institute, Av. Lineu Prestes, 2242-CEP Sao Paulo (Brazil); Collantes, Hugo D.C. [Chemical and Environmental Centre, Nuclear Energy Research Institute, Av. Lineu Prestes, 2242-CEP Sao Paulo (Brazil); Lugao, Ademar B. [Chemical and Environmental Centre, Nuclear Energy Research Institute, Av. Lineu Prestes, 2242-CEP Sao Paulo (Brazil)

    2005-07-01

    The type I allergy associated with the use of natural rubber latex (NRL) products is caused by the NRL proteins leached by the sweat or other body fluids. Makuuchi's group proposed for the first time the proteins removal by the addition of water-soluble polymers (WSP) on radiation vulcanization of natural rubber latex (RVNRL) that is a promising process under development in many countries. In this study, Brazilian field natural rubber was irradiated with a {sup 60}Co gamma source to reduce the content of WSP in the final product. WSP was used as additive to improve the extraction of protein. After irradiation the RVNRL was centrifuged to extract the WSP and proteins. The analytical methodology for protein content was based on the modified Lowry method according to ASTM D5712. Protein determination was carried out in serum of latex and in the extracts of the gloves. The concentration of extractable water-soluble proteins in serum of irradiated field NRL (NRL1), not irradiated one (NRL2); of twice centrifuged sample with polymer additive NRL (NRL3) and of the glove manufactured (NRLG) are compared with commercial glove (CG). The irradiation process increases the extractable water-soluble proteins, EP, as reported in the literature. In this study the use of polymeric additive on the bi-centrifugation process to remove protein was successful and the EP of the glove obtained in NRL3 was at around 40% of the commercial glove.

  19. Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry.

    Science.gov (United States)

    Tiong, Hung King; Hartson, Steven; Muriana, Peter M

    2015-03-01

    Extracts of surface proteins, with minimal artifacts from contaminating cytosolic components, are highly desirable for investigating surface factors involved in the attachment and formation of biofilms by bacteria that are problematic in commercial food processing facilities. In this study, we compared the protein profiles of the food pathogen, Listeria monocytogenes, recovered after applying different surface protein extraction methods compiled from the literature: trypsin-enzymatic shaving with BICAM/sucrose or Tris/sucrose buffers (Tryp B+S, Tryp T+S), Tris-buffered urea (UB), lithium chloride (LiCl) and Tris-buffered urea applied with hypotonic-stressed cells (UB-Ghost), and subjected them to liquid chromatography tandem mass spectrometry and protein identification. The data indicate that the UB-Ghost extraction method provides a cleaner extract of surface proteins including the predicted (this study and the literature) or validated members (literature) from L. monocytogenes. This was determined by an accumulative lower unique peptide number exhibited by mass spectrometry for total cytoplasmic proteins among different surface extracts, with a majority of proteins demonstrating hydrophilic properties. The extracted proteins were from different functional categories and have associations with the cell surface, intermediary metabolism, information pathways, or functionally unknown proteins as suggested by in silico analyses performed by other groups (Leger and ListiList). The utilization of an optimized method for surface protein extraction should greatly facilitate identification by LC-MS/MS that could be useful to anyone working on molecular proteomics of bacterial surfaces.

  20. Extraction of intracellular protein from Glaciozyma antarctica for proteomics analysis

    Science.gov (United States)

    Faizura, S. Nor; Farahayu, K.; Faizal, A. B. Mohd; Asmahani, A. A. S.; Amir, R.; Nazalan, N.; Diba, A. B. Farah; Muhammad, M. Nor; Munir, A. M. Abdul

    2013-11-01

    Two preparation methods of crude extracts of psychrophilic yeast Glaciozyma antarctica were compared in order to obtain a good recovery of intracellular proteins. Extraction with mechanical procedures using sonication was found to be more effective for obtaining good yield compare to alkaline treatment method. The procedure is simple, rapid, and produce better yield. A total of 52 proteins were identified by combining both extraction methods. Most of the proteins identified in this study involves in the metabolic process including glycolysis pathway, pentose phosphate pathway, pyruyate decarboxylation and also urea cyle. Several chaperons were identified including probable cpr1-cyclophilin (peptidylprolyl isomerase), macrolide-binding protein fkbp12 and heat shock proteins which were postulate to accelerate proper protein folding. Characteristic of the fundamental cellular processes inferred from the expressed-proteome highlight the evolutionary and functional complexity existing in this domain of life.

  1. Labile proteins accumulated in damaged hair upon permanent waving and bleaching treatments.

    Science.gov (United States)

    Inoue, Takafumi; Ito, Mayumi; Kizawa, Kenji

    2002-01-01

    We previously found that certain hair proteins were soluble by means of a partial extraction method. In this study, we demonstrate that the amount of soluble proteins internally formed in permed and bleached hair, labile proteins, is a useful index for hair damage assessment. Compared to tensile property changes, this index rose in widely dynamic ranges as the time of either permanent waving or bleaching treatments increased. The amount of labile proteins was much larger than that of proteins eluted into perming and bleaching lotions. However, the labile proteins showed electrophoretic profiles similar to those of the eluted proteins. These results suggest that a portion of the stable proteins in normal hair was transformed into labile proteins upon permanent waving and bleaching treatments. Consequently, permed and bleached hair tends to release the resultant labile proteins.

  2. comparison of protein extraction methods for the leaves of ficus ...

    African Journals Online (AJOL)

    F. I. Abdullah

    2017-05-01

    May 1, 2017 ... This study investigated several extraction methods for proteins from the leaves of Ficus .... found to be effective to remove plant lipids and pigments [22]. ... The addition of SDS as a solubilizing agent in the Tris buffered phenol.

  3. Optimal extraction and hydrolysis of Chlorella pyrenoidosa proteins.

    Science.gov (United States)

    Wang, Xiaoqin; Zhang, Xuewu

    2012-12-01

    In this study, for the first time, the applications of two new methods, ionic liquid and low-temperature high-pressure cell breakage methods, to the extraction of whole proteins in Chlorella pyrenoidosa cells were explored. Meanwhile, the comparison with three traditional methods was also made. The results indicated that the extraction rate for ionic liquid is only at moderate level, but the new low-temperature high-pressure cell breakage method can obviously increase the protein extraction rate up to 2- to 15-fold. Subsequently, the hydrolysis of the extracted proteins was conducted with three enzymes (papain, trypsin and alcalase). The data presented that the degree of hydrolysis for each enzyme under the optimal conditions is in the order of: alcalase (18.31%)>papain (14.33%)>trypsin (8.47%), demonstrating the potential of C. pyrenoidosa protein hydrolysates obtained here in nutritional supplement and medical foods.

  4. Techno-economical evaluation of protein extraction for microalgae biorefinery

    Science.gov (United States)

    Sari, Y. W.; Sanders, J. P. M.; Bruins, M. E.

    2016-01-01

    Due to scarcity of fossil feedstocks, there is an increasing demand for biobased fuels. Microalgae are considered as promising biobased feedstocks. However, microalgae based fuels are not yet produced at large scale at present. Applying biorefinery, not only for oil, but also for other components, such as carbohydrates and protein, may lead to the sustainable and economical microalgae-based fuels. This paper discusses two relatively mild conditions for microalgal protein extraction, based on alkali and enzymes. Green microalgae (Chlorella fusca) with and without prior lipid removal were used as feedstocks. Under mild conditions, more protein could be extracted using proteases, with the highest yields for microalgae meal (without lipids). The data on protein extraction yields were used to calculate the costs for producing 1 ton of microalgal protein. The processing cost for the alkaline method was € 2448 /ton protein. Enzymatic method performed better from an economic point of view with € 1367 /ton protein on processing costs. However, this is still far from industrially feasible. For both extraction methods, biomass cost per ton of produced product were high. A higher protein extraction yield can partially solve this problem, lowering processing cost to €620 and 1180 /ton protein product, using alkali and enzyme, respectively. Although alkaline method has lower processing cost, optimization appears to be better achievable using enzymes. If the enzymatic method can be optimized by lowering the amount of alkali added, leading to processing cost of € 633/ton protein product. Higher revenue can be generated when the residue after protein extraction can be sold as fuel, or better as a highly digestible feed for cattle.

  5. Extraction and fractionation of wheat flour proteins

    NARCIS (Netherlands)

    Graveland, A.; Bosveld, P.; Lichtendonk, W.J.; Moonen, H.H.E.; Scheepstra, A.

    1982-01-01

    Extraction of wheat flour with 1.5% sodium dodecyl sulphate (SDS) solution dissolved 65–67% of the total flour nitrogen. The SDS‐insoluble proteinaceous material was separated into glycoproteins‐I, II and III by ultracentrifugation. Part of the SDS‐soluble proteinaceous material was precipitated by

  6. Dietary Protein Affects Gene Expression and Prevents Lipid Accumulation in the Liver in Mice

    NARCIS (Netherlands)

    Schwarz, J.; Tome, D.G.; Baars, A.; Hooiveld, G.J.E.J.; Müller, M.R.

    2012-01-01

    Background and Aims: High protein (HP) diets are suggested to positively modulate obesity and associated increased prevalence of non-alcoholic fatty liver (NAFLD) disease in humans and rodents. The aim of our study was to detect mechanisms by which a HP diet affects hepatic lipid accumulation. Metho

  7. Accumulation of p53 protein in normal, dysplastic, and neoplastic Barrett's oesophagus

    NARCIS (Netherlands)

    K.K. Krishnadath (Kausilia); H.W. Tilanus (Hugo); M. van Blankenstein (Mark); F.T.B. Bosman (Fré); A.H. Mulder (Andries)

    1995-01-01

    textabstractAccumulation of p53 protein was determined by immunohistochemisty in archival material of biopsy specimens from 102 patients with Barrett's oesophagus with different grades of dysplasia, in 24 oesophageal adenocarcinomas associated with Barrett's oesophagus, and in 23 cases of metaplatic

  8. Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

    Science.gov (United States)

    Maruyama, Nobuyuki; Fujiwara, Keigo; Yokoyama, Kazunori; Cabanos, Cerrone; Hasegawa, Hisakazu; Takagi, Kyoko; Nishizawa, Keito; Uki, Yuriko; Kawarabayashi, Takeshi; Shouji, Mikio; Ishimoto, Masao; Terakawa, Teruhiko

    2014-10-01

    There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

  9. Comparison of Protein Extracts from Various Unicellular Green Sources

    Science.gov (United States)

    2017-01-01

    Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation techniques, proteins were extracted and isolated from four different unicellular sources (Arthrospira (spirulina) maxima, Nannochloropsis gaditana, Tetraselmis impellucida, and Scenedesmus dimorphus). Despite differences in protein contents of the sources (27–62% w/w) and in protein extractability (17–74% w/w), final protein isolates were obtained that had similar protein contents (62–77% w/w) and protein yields (3–9% w/w). Protein solubility as a function of pH was different between the sources and in ionic strength dependency, especially at pH < 4.0. Overall, the characterization and extraction protocol used allows a relatively fast and well-described isolation of purified proteins from novel protein sources. PMID:28701042

  10. 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

    2006-01-01

    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.

  11. Enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals

    NARCIS (Netherlands)

    Sari, Y.W.; Bruins, M.E.; Sanders, J.P.M.

    2013-01-01

    Oilseed meals that are by-products from oil production are potential resources for protein. The aim of this work is to investigate the use of enzymes in assisting in the extraction of protein from different oilseed meals, namely rapeseed, soybean, and microalgae meals. In addition, microalgae withou

  12. Enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals

    NARCIS (Netherlands)

    Sari, Y.W.; Bruins, M.E.; Sanders, J.P.M.

    2013-01-01

    Oilseed meals that are by-products from oil production are potential resources for protein. The aim of this work is to investigate the use of enzymes in assisting in the extraction of protein from different oilseed meals, namely rapeseed, soybean, and microalgae meals. In addition, microalgae withou

  13. Enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals

    NARCIS (Netherlands)

    Sari, Y.W.; Bruins, M.E.; Sanders, J.P.M.

    2013-01-01

    Oilseed meals that are by-products from oil production are potential resources for protein. The aim of this work is to investigate the use of enzymes in assisting in the extraction of protein from different oilseed meals, namely rapeseed, soybean, and microalgae meals. In addition, microalgae

  14. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

    DEFF Research Database (Denmark)

    Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

    2007-01-01

    Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae......) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen...... from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both...

  15. SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation.

    Science.gov (United States)

    O'Rourke, Jacqueline Gire; Gareau, Jaclyn R; Ochaba, Joseph; Song, Wan; Raskó, Tamás; Reverter, David; Lee, John; Monteys, Alex Mas; Pallos, Judit; Mee, Lisa; Vashishtha, Malini; Apostol, Barbara L; Nicholson, Thomas Peter; Illes, Katalin; Zhu, Ya-Zhen; Dasso, Mary; Bates, Gillian P; Difiglia, Marian; Davidson, Beverly; Wanker, Erich E; Marsh, J Lawrence; Lima, Christopher D; Steffan, Joan S; Thompson, Leslie M

    2013-07-25

    A key feature in Huntington disease (HD) is the accumulation of mutant Huntingtin (HTT) protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  16. SUMO-2 and PIAS1 Modulate Insoluble Mutant Huntingtin Protein Accumulation

    Directory of Open Access Journals (Sweden)

    Jacqueline Gire O’Rourke

    2013-07-01

    Full Text Available A key feature in Huntington disease (HD is the accumulation of mutant Huntingtin (HTT protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  17. The protein and lipid composition of arterial elastin and its relationship to lipid accumulation in the atherosclerotic plaque.

    Science.gov (United States)

    Kramsch, D M; Franzblau, C; Hollander, W

    1971-08-01

    Elastin preparations from intimal layers and the media of normal and atherosclerotic human aortae were analyzed for protein and lipid content. In atherosclerotic aortae, elastin from plaques was compared with elastin from adjacent normal appearing areas of the same aorta. Arterial elastin purified by alkaline extraction appeared to be a protein-lipid complex containing free and ester cholesterol, phospholipids, and triglycerides. The lipid component of normal arterial elastin was small (1-2%). With increasing severity of atherosclerosis, there was a progressive accumulation of lipid in intimal elastin from plaques, reaching a mean lipid content of 37% in severe plaques. The increase in the lipid content of plaque elastic preparations was mainly due to large increases in cholesterol, over 80% of which was cholesteryl ester. This deposition of cholesterol in plaque elastin accounted for 20-34% of the total cholesterol content of the plaque. The increased lipid deposition in plaque elastin was associated with alterations in the amino acid composition of plaque elastin. In elastin from plaque intima, the following polar amino acids were increased significantly: aspartic acid, threonine, serine, glutamic acid, lysine, histidine, and arginine; whereas, cross-linking amino acids: desmosine, isodesmosine, and lysinonorleucine were decreased significantly. The amino acid and lipid composition of elastin from normal appearing aortic areas was comparable to that of normal arterial elastin except for intimal elastin directly adjacent to and medial elastin directly below the most severe plaques.The data indicate that the focal lipid deposition in early atherosclerotic plaques is due to a large extent to lipid accumulations in altered elastin protein of localized intimal areas. Continued lipid deposition in altered elastin appears to contribute substantially to the progressive lipid accumulation in the plaque. The study suggests that elastin of intimal elastic membranes may play

  18. Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Chourey, Karuna [ORNL; Jansson, Janet [Lawrence Berkeley National Laboratory (LBNL); Verberkmoes, Nathan C [ORNL; Shah, Manesh B [ORNL; Chavarria, Krystle L. [Lawrence Berkeley National Laboratory (LBNL); Tom, Lauren M [Lawrence Berkeley National Laboratory (LBNL); Brodie, Eoin L. [Lawrence Berkeley National Laboratory (LBNL); Hettich, Robert {Bob} L [ORNL

    2010-01-01

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  19. Direct cellular lysis/protein extraction protocol for soil metaproteomics.

    Science.gov (United States)

    Chourey, Karuna; Jansson, Janet; VerBerkmoes, Nathan; Shah, Manesh; Chavarria, Krystle L; Tom, Lauren M; Brodie, Eoin L; Hettich, Robert L

    2010-12-03

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  20. Nutritional quality of sunflower seed protein fraction extracted with isopropanol.

    Science.gov (United States)

    Sen, M; Bhattacharyya, D K

    2000-01-01

    This study investigated the nutritional effect of sunflower seed protein fraction (SSPF) extracted with isopropanol on growth, plasma and tissue lipid profile, protein content and erythrocyte membrane lipid profile of rats. Dehulled sunflower seeds were extracted with isopropanol at 50 +/- 1 degree C resulting in a protein fraction (71.5%) with low residual chlorogenic acid (0.07%) and fiber (3.3%) contents. Rats fed the sunflower seed protein fraction had a similar body weight gain and food efficiency ratios in comparison to those fed casein. Rats fed SSPF in contrast had a significantly higher growth and food efficiency ratio than the rats fed sunflower meal (SM), extracted with hexane. However, dietary proteins exerted a separate effect on plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, low density lipoprotein to high density lipoprotein cholesterol (LDL-C/HDL-C) ratio and triglyceride content. Sunflower seed protein fraction resulted in a significant decrease in plasma cholesterol (p < 0.05) and LDL-cholesterol (p < 0.02) levels compared to the casein fed rats. Membrane phospholipid profile also showed a marked variation with the type of dietary protein. Rats fed SSPF and SM did not show much variation in plasma lipids, plasma proteins, liver and brain lipids and membrane phospholipid concentrations. Protein content, liver and brain lipid profile of the groups fed SSPF and casein were comparable, suggesting that the nutritional value of SSPF is better than SM and equivalent to that of casein.

  1. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins.

    Science.gov (United States)

    Sehnke, P C; Chung, H J; Wu, K; Ferl, R J

    2001-01-16

    In higher plants the production of starch is orchestrated by chloroplast-localized biosynthetic enzymes, namely starch synthases, ADP-glucose pyrophosphorylase, and starch branching and debranching enzymes. Diurnal regulation of these enzymes, as well as starch-degrading enzymes, influences both the levels and composition of starch, and is dependent in some instances upon phosphorylation-linked regulation. The phosphoserine/threonine-binding 14-3-3 proteins participate in environmentally responsive phosphorylation-related regulatory functions in plants, and as such are potentially involved in starch regulation. We report here that reduction of the epsilon subgroup of Arabidopsis 14-3-3 proteins by antisense technology resulted in a 2- to 4-fold increase in leaf starch accumulation. Dark-governed starch breakdown was unaffected in these "antisense plants," indicating an unaltered starch-degradation pathway and suggesting a role for 14-3-3 proteins in regulation of starch synthesis. Absorption spectra and gelatinization properties indicate that the starch from the antisense plants has an altered branched glucan composition. Biochemical characterization of protease-treated starch granules from both Arabidopsis leaves and maize endosperm showed that 14-3-3 proteins are internal intrinsic granule proteins. These data suggest a direct role for 14-3-3 proteins in starch accumulation. The starch synthase III family is a possible target for 14-3-3 protein regulation because, uniquely among plastid-localized starch metabolic enzymes, all members of the family contain the conserved 14-3-3 protein phosphoserine/threonine-binding consensus motif. This possibility is strengthened by immunocapture using antibodies to DU1, a maize starch synthase III family member, and direct interaction with biotinylated 14-3-3 protein, both of which demonstrated an association between 14-3-3 proteins and DU1 or DU1-like proteins.

  2. Two outer membrane proteins contribute to cellular fitness in Caulobacter crescentus by preventing intracellular S-layer protein accumulation.

    Science.gov (United States)

    Overton, K Wesley; Park, Dan M; Yung, Mimi C; Dohnalkova, Alice C; Smit, John; Jiao, Yongqin

    2016-09-23

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport S-layer protein from the cytoplasm to the cell exterior. Caulobacter crescentus is unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFa and RsaFb, that, together with other components, form a type I protein translocation pathway for S-layer export. These proteins have homology to E. coli TolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFa and RsaFb led to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and RNA-seq, we show that loss of both RsaFa and RsaFb led to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein mis-folding and degradation pathways. These findings provide new insight into the requirement for RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in C. crescentus IMPORTANCE: Decreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell largely due to lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural

  3. Panicovirus accumulation is governed by two membrane-associated proteins with a newly identified conserved motif that contributes to pathogenicity

    Directory of Open Access Journals (Sweden)

    Turina Massimo

    2006-03-01

    Full Text Available Abstract Panicum mosaic virus (PMV has a positive-sense, single-stranded RNA genome that serves as the mRNA for two 5'-proximal genes, p48 and p112. The p112 open reading frame (ORF has a GDD-motif, a feature of virus RNA-dependent RNA polymerases. Replication assays in protoplasts showed that p48 and p112 are sufficient for replication of PMV and its satellite virus (SPMV. Differential centrifugation of extracts from PMV-infected plants showed that the p48 and p112 proteins are membrane-associated. The same fractions exhibited RNA polymerase activity in vitro on viral RNA templates, suggesting that p48 and p112 represent the viral replication proteins. Moreover, we identified a domain spanning amino acids 306 to 405 on the p48 and p112 PMV ORFs that is common to the Tombusviridae. Alanine scanning mutagenesis of the conserved domain (CD revealed that several substitutions were lethal or severely debilitated PMV accumulation. Other substitutions did not affect RNA accumulation, yet they caused variable phenotypes suggestive of plant-dependent effects on systemic invasion and symptom induction. The mutants that were most debilitating to PMV replication were hydrophobic amino acids that we hypothesize are important for membrane localization and functional replicase activity.

  4. Extractable protein of radiation vulcanized natural rubber latex

    Energy Technology Data Exchange (ETDEWEB)

    Soebianto, Y.S. [Center for Research and Development of Isotopes and Radiation Technology, BATAN, Jakarta (Indonesia); Upul, R.M. [Rubber Research Institute of Sri Lanka, Ratmalana (Sri Lanka); Makuuchi, K.; Yoshii, F.; Kume, T. [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2000-03-01

    A new method to reduce the protein level in the latex products by irradiation is reported. Water soluble protein (WSP) solution (10%) was added into radiation vulcanized NR latex (RVNRL) as much as 3 phr in three different processes: added to RVNRL, added to re-centrifuged RVNRL, and added to RVNRL followed by centrifugation. The protein content was determined by enhanced BCA method, and identified by SDS-PAGE analysis. Addition of WSP followed by centrifugation reduces EP up to the minimum protein detection, and shortens the leaching time to 20-30 min. SDS-PAGE analysis confirms the reduction of soluble protein in the serum phase, and disappearance of protein bands in the rubber extract. Protein-WSP interaction produces water soluble complex, and removed by centrifugation. The molecular weight of WSP dictates the efficiency of protein removal. (author)

  5. Towards plant protein refinery: Review on protein extraction using alkali and potential enzymatic assistance.

    Science.gov (United States)

    Sari, Yessie W; Mulder, Wilhelmus J; Sanders, Johan P M; Bruins, Marieke E

    2015-08-01

    The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein extraction including the potentials of enzyme addition in the form of proteases and/or carbohydrolases. Strategic biomass selection, combined with the appropriate process conditions can increase protein yields after extraction. Enzyme addition, especially of proteases, can be useful when alkaline protein extraction yields are low. These additions can also be used to enable processing at a pH closer to 7 to avoid the otherwise severe conditions that denature proteins. Finally, a protein biorefinery concept is presented that aims to upcycle residual biomass by separating essential amino acids to be used for food and feed, and non-essential amino acids for production of bulk chemicals.

  6. Proteomic analysis identifies proteins related to carotenoid accumulation in Yesso scallop (Patinopecten yessoensis).

    Science.gov (United States)

    Zhang, Yueyue; Zhang, Lingling; Sun, Jin; Qiu, Jianwen; Hu, Xiaoli; Hu, Jingjie; Bao, Zhenmin

    2014-03-15

    Carotenoids are powerful antioxidants that affect many physiological functions. As an important source of natural carotenoids, marine mollusks contain various types of carotenoids and are receiving increasing research attention. To better understand the molecular mechanism underlying carotenoid accumulation in marine mollusks, a new variety of carotenoid-enriched Yesso scallop (Patinopecten yessoensis), named "Haida golden scallop", was used in this study. A proteomic approach was applied to explore the differences between the new variety and common individuals, resulting in seven differentially expressed proteins. Real-time PCR showed that four of the corresponding genes were also significantly up-regulated at the mRNA level in the new variety. Genes involved in various biological processes, such as lipid and glucose metabolism, protein-folding and degradation, were altered. Peroxisome proliferator-activated receptors (PPARs) may play a vital role in these changes. This study represents the first step towards future work on the genetic basis of carotenoid accumulation in marine mollusks.

  7. Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.

    Directory of Open Access Journals (Sweden)

    Sharifi Golandam

    2012-01-01

    Full Text Available Abstract Background Somatic embryogenesis (SE is a complex biological process that occurs under inductive conditions and causes fully differentiated cells to be reprogrammed to an embryo like state. In order to get a better insight about molecular basis of the SE in Crocus sativus L. and to characterize differentially accumulated proteins during the process, a proteomic study based on two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry has been carried out. Results We have compared proteome profiles of non-embryogenic and embryogenic calli with native corm explants. Total soluble proteins were phenol-extracted and loaded on 18 cm IPG strips for the first dimension and 11.5% sodium dodecyl sulfate-polyacrylamide gels for the second dimension. Fifty spots with more than 1.5-fold change in abundance were subjected to mass spectrometry analysis for further characterization. Among them 36 proteins could be identified, which are classified into defense and stress response, protein synthesis and processing, carbohydrate and energy metabolism, secondary metabolism, and nitrogen metabolism. Conclusion Our results showed that diverse cellular and molecular processes were affected during somatic to embryogenic transition. Differential proteomic analysis suggests a key role for ascorbate metabolism during early stage of SE, and points to the possible role of ascorbate-glutathione cycle in establishing somatic embryos.

  8. Screening of Argentine plant extracts: impact on growth parameters and aflatoxin B1 accumulation by Aspergillus section Flavi.

    Science.gov (United States)

    Bluma, R; Amaiden, M R; Etcheverry, M

    2008-02-29

    The effect of essential oils, ethanolic and aqueous extract of 41 vegetable species on Aspergillus section Flavi growth was evaluated. The in vitro screen was a two-stage process. A wide-spectrum initial screen which identified promising antifungal plant extracts was carried out first. After that, identified extracts were studied in more detail by in vitro assays. A total of 96 plant extracts were screened. Essential oils were found to be the most effective extract controlling aflatoxigenic strains. Clove, mountain thyme, poleo and eucalyptus essential oils were selected to study their antifungal effect. Studies on percentage of germination, germ-tube elongation rate, growth rate, and aflatoxin B1 accumulation were carried out. Clove, mountain thyme and poleo essential oils showed the most antifungal effect under all growth parameters analyzed as well as aflatoxin B1 accumulation. Our results suggest that mountain thyme and poleo, which are native vegetal species of Argentina, and clove essential oils could be used alone or in conjunction with other substances to control the presence of aflatoxigenic fungi in stored maize.

  9. A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation

    OpenAIRE

    Lubomir N. Sokolov; Dominguez-Solis, Jose R.; Allary, Anne-Laure; Buchanan, Bob B.; Luan, Sheng

    2006-01-01

    Starch is the ultimate storage molecule formed in the photosynthetic fixation of carbon dioxide by chloroplasts. Starch accumulates during the day and is degraded at night to intermediates that are exported to heterotrophic organs. The mechanism by which diurnal cycles control the transitory biosynthesis and degradation of chloroplast starch has long remained a mystery. We now report evidence that a dual-specificity protein phosphatase, DSP4, binds to starch granules during the day and dissoc...

  10. Influence of host chloroplast proteins on Tobacco mosaic virus accumulation and intercellular movement.

    Science.gov (United States)

    Bhat, Sumana; Folimonova, Svetlana Y; Cole, Anthony B; Ballard, Kimberly D; Lei, Zhentian; Watson, Bonnie S; Sumner, Lloyd W; Nelson, Richard S

    2013-01-01

    Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase- and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase γ-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pull-down analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca- or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC- and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front.

  11. Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

    Science.gov (United States)

    Nitta, Yoko; Yasukata, Fumiko; Kitamoto, Noritoshi; Ito, Mikiko; Sakaue, Motoyoshi; Kikuzaki, Hiroe; Ueno, Hiroshi

    2016-03-01

    Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

  12. A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins.

    Science.gov (United States)

    Cilia, M; Fish, T; Yang, X; McLaughlin, M; Thannhauser, T W; Gray, S

    2009-09-01

    Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.

  13. Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein

    Energy Technology Data Exchange (ETDEWEB)

    Sekine, Shuichi; Ito, Konomi; Watanabe, Haruna; Nakano, Takafumi [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan); Moriya, Kyoji; Shintani, Yoshizumi; Fujie, Hajime; Tsutsumi, Takeya; Miyoshi, Hideyuki; Fujinaga, Hidetake; Shinzawa, Seiko; Koike, Kazuhiko [Department of Internal Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Horie, Toshiharu, E-mail: t.horie@thu.ac.jp [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan)

    2015-02-01

    Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ({sup 59}Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca{sup 2+} uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein. - Highlights: • Iron accumulation in the livers of patients with hepatitis C virus (HCV) infection is thought to exacerbate oxidative stress. • The impact of iron overload on mitochondrial damage and ROS production in HCV core protein-expressing cells were examined. • Mitochondrial iron uptake was increased in the livers of HCV core protein-expressing transgenic mice. • Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates

  14. Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani.

    Science.gov (United States)

    Lakshman, Dilip K; Natarajan, Savithiry S; Lakshman, Sukla; Garrett, Wesley M; Dhar, Arun K

    2008-01-01

    Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris, T. praticola) is a basidiomycetous fungus and a major cause of root diseases of economically important plants. Various isolates of this fungus are also beneficially associated with orchids, may serve as biocontrol agents or remain as saprophytes with roles in decaying and recycling of soil organic matter. R. solani displays several hyphal anastomosis groups (AG) with distinct host and pathogenic specializations. Even though there are reports on the physiological and histological basis of Rhizoctonia-host interactions, very little is known about the molecular biology and control of gene expression early during infection by this pathogen. Proteamic technologies are powerful tools for examining alterations in protein profiles. To aid studies on its biology and host pathogen interactions, a two-dimensional (2-D) gel-based global proteomic study has been initiated. To develop an optimized protein extraction protocol for R. solani, we compared two previously reported protein extraction protocols for 2-D gel analysis of R. solani (AG-4) isolate Rs23. Both TCA-acetone precipitation and phosphate solubilization before TCA-acetone precipitation worked well for R. solani protein extraction, although selective enrichment of some proteins was noted with either method. About 450 spots could be detected with the densitiometric tracing of Coomassie blue-stained 2-D PAGE gels covering pH 4-7 and 6.5-205 kDa. Selected protein spots were subjected to mass spectrometric analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Eleven protein spots were positively identified based on peptide mass fingerprinting match with fungal proteins in public databases with the Mascot search engine. These results testify to the suitability of the two optimized protein extraction protocols for 2-D proteomic studies of R. solani.

  15. [Extraction and application of Perna viridis foot protein as bioadhesive].

    Science.gov (United States)

    Jiang, Zhen; Liu, Jiapeng; Jin, Lihua; Zhang, Qiqing

    2010-12-01

    Mussel adhesive proteins have attracted increasing interests for their potential use as environmentally friendly bioadhesives in medicine and aqueous conditions. In this study, surface coating analysis, quartz crystal microbalance (QCM), cell and bone tissue adhesion and cytotoxicity assay were used to study the properties of the Perna viridis foot proteins (Pvfp) extract as bioadhesive. The results of coating ability on various materials and QCM analysis revealed that Pvfp extract has comparable or superior adsorbtion ability to that of Cell-Tak (the naturally extracted MAP mixture from Mytilus edulis, and has been commercialized), and also, the cell adhesion ability of Pvfp extract was stronger than that of Cell-Tak and poly-L-lysine. No cytotoxicity was detected using human HeLa and 293T cells. Furthermore, broken bones of mouse could be stuck together by use of Pvfp extract. In bulk-scale adhesion tests, Pvfp extract showed much greater tensile strength than did fibrin glue for conglutinating poly (vinl chloride) sticks and for binding together pig's femur segments. These results suggested that Pvfp extract be an efficient cell and tissue adhesive in biotechnological application and it might be a potential bioadhesive in medical practice.

  16. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

  17. Cytochrome P4501A induction and porphyrin accumulation in PLHC-1 fish cells exposed to sediment and oil shale extracts.

    Science.gov (United States)

    Huuskonen, S E; Tuvikene, A; Trapido, M; Fent, K; Hahn, M E

    2000-01-01

    The present study describes the use of a fish hepatoma cell line (PLHC-1) in monitoring the biological effects of sediments collected from recipient waters of the oil shale industry. Sampling sites were located in River Purtse and River Kohtla in northeast Estonia. The effects of pure oil shale on the PLHC-1 cells were also studied. The cells were exposed to n-hexane-extracted samples in 48-well plates for 24 h, and 7-ethoxyresorufin O-deethylase (EROD) activity, total protein, and porphyrin content were measured in the exposed cells. Polycyclic aromatic hydrocarbon (PAH) contents in the samples were measured by high-performance liquid chromatography (HPLC). All the sediment and oil shale samples induced CYP1A activity and led to porphyrin accumulation in the cells. The most potent inducers were the sediments collected near the oil shale processing plants (site Lüganuse in River Purtse and Kohtla in River Kohtla), as well as those at the most downstream site in River Purtse (Purtse). These samples possessed high total PAH contents, ranging from 4,270 to nearly 150,000 microg/kg dry sediment. The presence of other lipophilic organic contaminants in the samples was not determined in this study. Both EROD activity and porphyrin content exhibited biphasic induction curves, and the ED(50)(1) values for EROD activity were lower than the ED(50)s for porphyrin content. 2,3,7, 8-Tetrachlorodibenzo-p-dioxin induction equivalents (TCDD-EQs) calculated from EROD induction potencies correlated well with total PAHs (r(2) = 0.827 and p = 0.003 for log-transformed data) and also with individual PAHs. TCDD-EQs for porphyrin content did not correlate significantly with total PAHs (log-log r(2) = 0.785, p = 0. 116). The biological potency and PAH contamination of the samples showed the same rank order, except at Lüganuse, where sediment extracts induced CYP1A and porphyrins more than could have been expected based on PAH contents. Bioassay-derived induction EQs (normalized to

  18. Correlation between protein accumulation profiles and conventional toxicological findings using a model antiandrogenic compound, flutamide.

    Science.gov (United States)

    Friry-Santini, Claire; Rouquié, David; Kennel, Philippe; Tinwell, Helen; Benahmed, Mohamed; Bars, Rémi

    2007-05-01

    In conventional rodent toxicity studies the characterization of the adverse effects of a chemical relies primarily on gravimetric, and histopathological data. The aim of this study was to evaluate if the use of two-dimensional gel electrophoresis could generate protein accumulation profiles, which were in accordance with conventional toxicological findings by investigating a model antiandrogen, flutamide (FM), whose toxic effects, as measured using standard approaches, are well characterized. Male Sprague-Dawley rats were orally exposed to FM (0, 6, 30, and 150 mg/kg/day) for 28 days. The expected inhibition of androgen-dependent tissue stimulation, increased luteinizing hormone and testosterone plasma levels, and Leydig cell hyperplasia were observed. Changes in testicular protein accumulation profiles were evaluated in rats exposed to 150 mg/kg/day FM. Several proteins involved in steroidogenesis (e.g., StAR, ApoE, Hmgcs1, Idi1), cell cycle, and cancer (e.g., Ddx1, Hspd1) were modulated by FM, and these data provided molecular evidence for the hormonal and testicular histopathology changes recorded. Changes in proteins associated with spermatogenesis were also recorded, and these are discussed within the context of the testicular phenotype observed following FM treatment (i.e., normal spermatogenesis but Leydig cell hyperplasia). Overall, our data indicate that the combination of conventional toxicology measurements with omic observations has the potential to improve our global understanding of the toxicity of a compound.

  19. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    Science.gov (United States)

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  20. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    Science.gov (United States)

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress.

  1. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

    DEFF Research Database (Denmark)

    Hunter, Roger W; Treebak, Jonas Thue; Wojtaszewski, Jørgen

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically......, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation. RESEARCH DESIGN...... AND METHODS We recently generated knock-in mice in which wild-type muscle GS was replaced by a mutant (Arg582Ala) that could not be activated by glucose-6-phosphate (G6P), but possessed full catalytic activity and could still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic...

  2. UCH-L1 induces podocyte hypertrophy in membranous nephropathy by protein accumulation.

    Science.gov (United States)

    Lohmann, Frithjof; Sachs, Marlies; Meyer, Tobias N; Sievert, Henning; Lindenmeyer, Maja T; Wiech, Thorsten; Cohen, Clemens D; Balabanov, Stefan; Stahl, R A K; Meyer-Schwesinger, Catherine

    2014-07-01

    Podocytes are terminally differentiated cells of the glomerular filtration barrier that react with hypertrophy in the course of injury such as in membranous nephropathy (MGN). The neuronal deubiquitinase ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed and activated in podocytes of human and rodent MGN. UCH-L1 regulates the mono-ubiquitin pool and induces accumulation of poly-ubiquitinated proteins in affected podocytes. Here, we investigated the role of UCH-L1 in podocyte hypertrophy and in the homeostasis of the hypertrophy associated "model protein" p27(Kip1). A better understanding of the basic mechanisms leading to podocyte hypertrophy is crucial for the development of specific therapies in MGN. In human and rat MGN, hypertrophic podocytes exhibited a simultaneous up-regulation of UCH-L1 and of cytoplasmic p27(Kip1) content. Functionally, inhibition of UCH-L1 activity and knockdown or inhibition of UCH-L1 attenuated podocyte hypertrophy by decreasing the total protein content in isolated glomeruli and in cultured podocytes. In contrast, UCH-L1 levels and activity increased podocyte hypertrophy and total protein content in culture, specifically of cytoplasmic p27(Kip1). UCH-L1 enhanced cytoplasmic p27(Kip1) levels by nuclear export and decreased poly-ubiquitination and proteasomal degradation of p27(Kip1). In parallel, UCH-L1 increased podocyte turnover, migration and cytoskeletal rearrangement, which are associated with known oncogenic functions of cytoplasmic p27(Kip1) in cancer. We propose that UCH-L1 induces podocyte hypertrophy in MGN by increasing the total protein content through altered degradation and accumulation of proteins such as p27(Kip1) in the cytoplasm of podocytes. Modification of both UCH-L1 activity and levels could be a new therapeutic avenue to podocyte hypertrophy in MGN. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Sequential extractions: A new way for protein quantification-data from peanut allergens.

    Science.gov (United States)

    Zhou, Ningling; Li, Wenying; Wu, Zhihua; Li, Xin; Yang, Anshu; Tong, Ping; Chen, Hongbing

    2015-09-01

    Quantification of certain protein contents in the matrix is essential in protein analyses. The amount of total protein in the matrix can be determined by the Kjeldahl method. However, few methods can quantify certain protein contents in the matrix without extracting all of them in solution. Extracting all of the contents is difficult for proteins, especially relatively insoluble ones. A five-step sequential extraction method was developed for the quantification of certain proteins in defatted peanut flour based on the relationship between the extracted protein contents and the extraction times. The extracted proteins (i.e., total protein, Ara h 1, and Ara h 2) were quantitatively analyzed in each extraction of the same condition. An exponential equation was obtained between the extraction times and the respective amount of extracted protein as well as both the total protein and a particular protein. In particular, the amount of protein extracted each time can be a geometric sequence. If all proteins can be extracted with sufficient extraction times, the protein contents in the peanut matrix can be calculated using a mathematical summation formula. This sum should be all proteins in the matrix. The five-step sequential extraction method can provide a means to quantify certain proteins in the matrix.

  4. A Shortest Dependency Path Based Convolutional Neural Network for Protein-Protein Relation Extraction

    OpenAIRE

    2016-01-01

    The state-of-the-art methods for protein-protein interaction (PPI) extraction are primarily based on kernel methods, and their performances strongly depend on the handcraft features. In this paper, we tackle PPI extraction by using convolutional neural networks (CNN) and propose a shortest dependency path based CNN (sdpCNN) model. The proposed method (1) only takes the sdp and word embedding as input and (2) could avoid bias from feature selection by using CNN. We performed experiments on sta...

  5. Recent Advances in Protein Extraction and Chiral Separation of Biomolecules

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Reverse micelles create unique environment in organic media. They are capable of solubilizing hydrophilic biomolecules (e.g., proteins, peptides, amino acids, and DNAs) in their aqueous interior. This feature brings about the practical use of biomaterials in organic media because reverse micelles solubilize them with the intrinsic activity. In this paper, we focus on recent two topics concerning protein extraction and chiral separation of biomolecules using liquid membranes. In the first topic, we present recent attempts to extract proteins from an aqueous solution into isooctane using reverse micelles, and some important operational parameters to achieve an efficient protein transfer are discussed. Furthermore, novel function of reverse micelles as a protein activation medium is introduced. In the reverse micellar phase, denatured proreins were completely reactivated in the reverse micellar solution. The reverse micellar technique is found to be a useful tool not only for protein separation but also for protein refolding. Furthermore, we found that a cyclic ligand carixarene has an extraction ability to set up optimum conditions for protein transfer. In the second topic, we have found that a supported liquid membrane (SLM) encapsulating enzymes shows high enantioselectivity (enantioselective excess value is over 96%) in the transport of racemic pharmaceutical compound ibuprofen. A different experiment also suggests that the α-chymotrypsin-catalyzed reactions droved the enantioselective transport of L-phenylalanine based on the enantioselectivity of the enzyme. The SLM encapsulating the surfactant-enzyme complex enabled the highly enantioselective separation of racemic mixtures. It can be envisioned that arrangement of appropriate enzymes in the SLM system will allow enantioselective separation of various useful organic compounds.

  6. Comparison of Nuclear Accumulation of p53 Protein with Mutations in the p53 Gene of Human Breast Cancer Tissues

    Institute of Scientific and Technical Information of China (English)

    王萱仪; 查小明; 武正炎; 范萍

    2001-01-01

    Objective The objective was to compare nuclear accumulation of p53 protein with mutations in the p53 gene on the tissues of human breast cancer. Methods Fifty-four invasive ductal carcinomas of breast were analyzed by the method of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) silver stain and strep-avidin-biotin-peroxidase complex (SABC) immunohistochemistry. Results A statistically significant association between the presence of p53 gene mutation and nuclear accumulation of p53 protein was found (P<0.01). 22 tumors that demonstrated p53 gene mutations showed nuclear accumulation of p53 protein, while only 9 (28%) showed nuclear accumulation of p53 protein in 32 tumors without p53 gene mutations. Both p53 mutation protein and p53 gene mutations were prevalent in steroid and progesterone receptors negative tumors (P<0.05). A statistically significant association was found between the nuclear accumulation of p53 protein and lymph node invasion (P<0.05), and between p53 gene mutations and lymph node invasion (P<0.05). p53 abnormalities might be associated with an aggressive phenotype in breast cancer. Conclusion The immunohistochemical detection of nuclear p53 protein accumulation is highly associated with p53 gene mutations in breast cancer tissues, and that this method is useful for rapid screening of p53 abnormalities. However, in order to avoid false positive reaction, the p53 gene mutations should be determined in cases slightly positive for p53 nuclear protein.

  7. Inhibitory effect of Piper betel leaf extracts on copper-mediated LDL oxidation and oxLDL-induced lipid accumulation via inducing reverse cholesterol transport in macrophages.

    Science.gov (United States)

    Ma, Gwo-Chin; Wu, Pei-Fang; Tseng, Hsien-Chun; Chyau, Charng-Cherng; Lu, Hsiu-Chin; Chou, Fen-Pi

    2013-12-15

    Piper betel leaf (PBL) has the biological capabilities of detoxification and can work as an anti-inflammatory agent and an anti-oxidant. In this study, we evaluated the anti-oxidative activity of the extract of Piper betel leaves (PBLs) on the basis of Cu(2+)-mediated oxidation, and its ability to prevent foam cell formation in a model for oxidised low density lipoprotein (oxLDL)-induced lipid accumulation in macrophages. Our data demonstrated that PBLs were able to inhibit LDL oxidation in vitro and are able to reduce the lipid accumulation in macrophages. We showed the underlying mechanisms to be the following: PBLs up-regulated the protein levels of the class A and class B scavenger receptors, the membrane lipid transporter ABCA1, and its upstream regulator Liver X receptor (LXR) in the macrophages exposed to oxLDL. The results suggested that PBLs activated the reverse cholesterol transport mechanism to enhance the metabolism of the oxLDL that could prevent both lipid accumulation and foam cell formation and further minimise the possible damage of vessels caused by the oxLDL.

  8. PRELIMINARY STUDY OF EXTRACTABLE PROTEIN BINDING USING MALEIC ANHYDRIDE COPOLYMER

    Institute of Scientific and Technical Information of China (English)

    Thirawan Nipithakul; Ladawan Watthanachote; Nanticha Kalapat

    2012-01-01

    A preliminary study of using maleic anhydride copolymer for protein binding has been carried out.The polymeric films were prepared by compression of the purified resin and annealing the film to induce efficient back formation of the anhydride groups.The properties of the film surface were analyzed by attenuated total reflection Fourier transforms infrared spectroscopy and water contact angle measurements.The protein content was determined by Bradford assay.To obtain optimum conditions,immersion time for protein binding was examined.Results revealed that proteins can be successfully immobilized onto the film surface via covalent linkage.The efficiency of the covalent binding of the extractable protein to maleic anhydride-polyethylene film was estimated at 69.87 μtg/cm2,although the film had low anhydride content (3%) on the surface.

  9. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice C.; Smit, John; Jiao, Yongqin; Parales, R. E.

    2016-09-23

    ABSTRACT

    Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

  10. Adverse effects of the renal accumulation of haem proteins. Novel therapeutic approaches.

    Science.gov (United States)

    Guerrero-Hue, Melania; Rubio-Navarro, Alfonso; Sevillano, Ángel; Yuste, Claudia; Gutiérrez, Eduardo; Palomino-Antolín, Alejandra; Román, Elena; Praga, Manuel; Egido, Jesús; Moreno, Juan Antonio

    2017-06-28

    Haemoglobin and myoglobin are haem proteins that play a key role as they help transport oxygen around the body. However, because of their chemical structure, these molecules can exert harmful effects when they are released massively into the bloodstream, as reported in certain pathological conditions associated with rhabdomyolysis or intravascular haemolysis. Once in the plasma, these haem proteins can be filtered and can accumulate in the kidney, where they become cytotoxic, particularly for the tubular epithelium, inducing acute kidney failure and chronic kidney disease. In this review, we will analyse the different pathological contexts that lead to the renal accumulation of these haem proteins, their relation to both acute and chronic loss of renal function, the pathophysiological mechanisms that cause adverse effects and the defence systems that counteract such actions. Finally, we will describe the different treatments currently used and present new therapeutic options based on the identification of new cellular and molecular targets, with particular emphasis on the numerous clinical trials that are currently ongoing. Copyright © 2017 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

  11. Proteins identified from care solution extractions of silicone hydrogels.

    Science.gov (United States)

    Emch, Andrew J; Nichols, Jason J

    2009-02-01

    The purpose of this study was to investigate the quantity and identify the proteins extracted from two different types of silicone hydrogel contact lenses by several multipurpose care solutions after 1 day of wear. Ten subjects were recruited to wear galyfilcon A lenses (Acuvue Advance, Vistakon) followed by lotrafilcon B lenses (O2 Optix, CIBA Vision) each for four consecutive days. Each day, subjects inserted a new pair of lenses for 8 h of wear after which both lenses were removed using forceps (lenses were not rubbed or rinsed after removal). Lenses were pooled in one of four commercially available care solutions for a 24-h soak followed by precipitation, resuspension in water, and quantification by Bradford assay and identification by mass spectrometry. Protein recovery from care solutions was as follows (quantities are in microg/lens): AQuify (galyfilcon A: 0.56, lotrafilcon B: 1.24), Complete MoisturePlus (galyfilcon A: 1.44, lotrafilcon B: 1.47), Opti-Free Express (galyfilcon A: 2.31, lotrafilcon B: 5.67), and ReNu MoistureLoc (galyfilcon A: 1.17, lotrafilcon B: 4.38). For each care solution, greater quantities of protein were removed from lotrafilcon B (3.19 +/- 2.19 microg/lens) than from galyfilcon A (1.37 +/- 0.72 microg/lens). Lactoferrin, lysozyme, and lipocalin were the most commonly identified, whereas various keratin compounds and other unique proteins were also detected. Opti-Free Express was consistently associated with the more efficient removal of proteins from these silicone hydrogels. More total protein was removed from lotrafilcon B than from galyfilcon A (approximately 2 x more protein) for all four care solutions, and 12 total unique protein species were recovered from galyfilcon A, whereas only 10 were recovered from lotrafilcon B. The higher quantities of protein extracted from lotrafilcon B may be due to stronger protein binding with this material and/or to differences in solution efficacy.

  12. Deep eutectic solvents as novel extraction media for protein partitioning.

    Science.gov (United States)

    Zeng, Qun; Wang, Yuzhi; Huang, Yanhua; Ding, Xueqin; Chen, Jing; Xu, Kaijia

    2014-05-21

    Four kinds of green deep eutectic solvent (DES) were synthesized, including choline chloride (ChCl)-urea, tetramethylammonium chloride (TMACl)-urea, tetrapropylammonium bromide (TPMBr)-urea and ChCl-methylurea. An aqueous two-phase system (ATPS) based ChCl-urea DES was studied for the first time for the extraction of bovine serum albumin (BSA). Single factor experiments proved that the extraction efficiency of BSA was influenced by the mass of the DES, concentration of K2HPO4 solution, separation time and extraction temperature. The optimum conditions were determined through an orthogonal experiment with the four factors described above. The results showed that under the optimum conditions, the average extraction efficiency could reach up to 99.94%, 99.72%, 100.05% and 100.05% (each measured three times). The relative standard deviations (RSD) of extraction efficiencies in precision, repeatability and stability experiments were 0.5533% (n = 5), 0.8306% (n = 5) and 0.9829% (n = 5), respectively. UV-vis and FT-IR spectra confirmed that there were no chemical interactions between BSA and the DES in the extraction process, and the CD spectra proved that the conformation of BSA did not change after extraction. The conductivity, DLS and TEM were combined to investigate the microstructure of the top phase and the possible mechanism for the extraction. The results showed that hydrophobic interactions, hydrogen bonding interactions and the salting-out effect played important roles in the transfer process, and the aggregation and surrounding phenomenon were the main driving forces for the separation. All of these results proved that ionic liquid (IL)-based ATPSs could potentially be substituted with DES-based ATPSs to offer new possibilities in the extraction of proteins.

  13. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    Science.gov (United States)

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.

  14. Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

    Science.gov (United States)

    Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Heide, Richard S Vander; Howard, Timothy; Everett, Allen D; Herrington, David; Van Eyk, Jennifer E

    2013-04-01

    Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Predicting the indicators of development of the Emba deposits with high visocity oils based on models of the flow rate and accumulated extraction

    Energy Technology Data Exchange (ETDEWEB)

    Dzhakiyev, K.T.

    1983-01-01

    Two types of curves of flow rate and accumulated extraction are identified on the basis of processing and analysis of actual data from the operation of the Emba deposits with increased and high viscosity oils. This made it possible to acquire two models of flow rate and accumulated extraction for deposits being developed in a water and pressure mode of mean activity and close to hard activity. An expression is acquired of the evaluation coefficient of extraction which is used in rating the annual (maximal) extraction and in designing a model of the flow rate and the accumulated extraction. A comparison of the actual curves of flow rate and accumulated extraction is given with models which are built based on an assigned volume of the extractable reserves and the parameters of the stratum and the fluids. A good convergence is noted between the calculated and factual curves of flow rate and accumulated extraction. The acquired models are recommended in rating the volumes of oil extraction by deposits which have left the stage of commercial exploration and by deposits which are entering the latter stage of development.

  16. Protein and Carbohydrate Accumulation in Normal and High-Lysine Barley in Spike Culture

    DEFF Research Database (Denmark)

    Mather, D.E; Giese, Nanna Henriette

    1984-01-01

    -soluble, hordein, and non-protein N fractions appeared to be normal. Endosperm dry weight and starch were lower than in intact plants and declined at higher N levels. A linear relationship was observed between starch content and the concentration of sucrose in the endosperm water. Uptake of culture medium......Spikes of barley cv. Bomi and high-lysine mutants Riso 1508 and Riso 56 were cultured on liquid media at varying N and sucrose levels. Bomi accumulated N in response to increasing N levels in the medium and a higher level was reached than in spikes of intact plants. The distribution of N in salt...

  17. Large-scale extraction of recombinant proteins from bacteria.

    Science.gov (United States)

    Simpson, Richard J

    2010-09-01

    Bacteria are particularly convenient for producing recombinant proteins for purification purposes. Suitable extraction methods for bacterial cells include sonication, glass bead milling, grinding with alumina or sand, high-pressure shearing using the French pressure cell (French Press), and lysozyme treatment. These procedures are applicable for preparing extracts from a variety of gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae, and gram-positive bacteria such as Bacillus subtilis. Disruption of bacterial cells by enzymatic means is commonly used because a relatively uniform treatment is obtained when cells are in suspension. This protocol describes the enzymatic disruption of E. coli.

  18. Fatty Acid Accumulation and Resulting PPARα Activation in Fibroblasts due to Trifunctional Protein Deficiency

    Directory of Open Access Journals (Sweden)

    Masato Wakabayashi

    2012-01-01

    Full Text Available To examine fatty acid accumulation and its toxic effects in cells, we analyzed skin fibroblasts from six patients with mitochondrial trifunctional protein deficiency, who had abnormalities in the second through fourth reactions in fatty acid β-oxidation system. We found free fatty acid accumulation, enhanced three acyl-CoA dehydrogenases, catalyzing the first reaction in the β-oxidation system and being assumed to have normal activities in these patients, and PPARα activation that was confirmed in the experiments using MK886, a PPARα specific antagonist and fenofibrate, a PPARα specific agonist. These novel findings suggest that the fatty acid accumulation and the resulting PPARα activation are major causes of the increase in the β-oxidation ability as probable compensation for fatty acid metabolism in the patients’ fibroblasts, and that enhanced cell proliferation and increased oxidative stress due to the PPARα activation relate to the development of specific clinical features such as hypertrophic cardiomyopathy, slight hepatomegaly, and skeletal myopathy. Additionally, significant suppression of the PPARα activation by means of MK886 treatment is assumed to provide a new method of treating this deficiency.

  19. The Components of Flemingia macrophylla Attenuate Amyloid β-Protein Accumulation by Regulating Amyloid β-Protein Metabolic Pathway

    Directory of Open Access Journals (Sweden)

    Yun-Lian Lin

    2012-01-01

    Full Text Available Flemingia macrophylla (Leguminosae is a popular traditional remedy used in Taiwan as anti-inflammatory, promoting blood circulation and antidiabetes agent. Recent study also suggested its neuroprotective activity against Alzheimer's disease. Therefore, the effects of F. macrophylla on Aβ production and degradation were studied. The effect of F. macrophylla on Aβ metabolism was detected using the cultured mouse neuroblastoma cells N2a transfected with human Swedish mutant APP (swAPP-N2a cells. The effects on Aβ degradation were evaluated on a cell-free system. An ELISA assay was applied to detect the level of Aβ1-40 and Aβ1-42. Western blots assay was employed to measure the levels of soluble amyloid precursor protein and insulin degrading enzyme (IDE. Three fractions of F. macrophylla modified Aβ accumulation by both inhibiting β-secretase and activating IDE. Three flavonoids modified Aβ accumulation by activating IDE. The activated IDE pool by the flavonoids was distinctly regulated by bacitracin (an IDE inhibitor. Furthermore, flavonoid 94-18-13 also modulates Aβ accumulation by enhancing IDE expression. In conclusion, the components of F. macrophylla possess the potential for developing new therapeutic drugs for Alzheimer's disease.

  20. Purification of matrix Gla protein from a marine teleost fish, Argyrosomus regius: calcified cartilage and not bone as the primary site of MGP accumulation in fish.

    Science.gov (United States)

    Simes, D C; Williamson, M K; Ortiz-Delgado, J B; Viegas, C S B; Price, P A; Cancela, M L

    2003-02-01

    Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5'rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was

  1. Lysosome dysfunction enhances oxidative stress-induced apoptosis through ubiquitinated protein accumulation in Hela cells.

    Science.gov (United States)

    Yu, Chunyan; Huang, Xiaowei; Xu, Ye; Li, Hongyan; Su, Jing; Zhong, Jiateng; Kang, Jinsong; Liu, Yuhe; Sun, Liankun

    2013-01-01

    The role of lysosomal system in oxidative stress-induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH(4)Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH(4)Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione-induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress.

  2. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

    DEFF Research Database (Denmark)

    Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

    2007-01-01

    that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved...... from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both...... floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows...

  3. Effect of Piper betel leaf stalk extract on protein metabolism in reproductive tissues of male albino rats

    Institute of Scientific and Technical Information of China (English)

    Vengaiah V; Govardhan Naik A; Changamma C

    2015-01-01

    Objective: To know the impact of Piper betel leaf stalk (P. betel) extract on Protein and energy metabolism and its role in male albino rats. Methods: Healthy adult (3-4 months old) male Wistar strain albino rats were administered with betel leaf stalk extract, at the dose of 50 mg/kg/day through oral gavages for 15 days. Twenty four hours after the last dose, the animals were autopsied. In order to assess antifertility effect in testis, epididymis, seminal vesicle and prostate gland, estimation of total, soluble and structural proteins, free amino acids and DNA, RNA were undertaken. Results: The accumulation in proteins indicates the anti-androgenic effect of extract. The reduction in free amino acids will affect the sertoli cell function, results in the damage of spermatogenesis. The significant elevation in testicular DNA content (hyperplasia) was observed. In the present study, P. betel leaf stalk extract decreases the concentration of RNA, in testes, seminal vesicle and prostate gland except in epididymis where it was elevated. It indicates the alterations in rate of protein synthesis and growth rate of tissues due to the administration of P. betel leaf stalk extraction. However, the RNA: DNA ratio was reduced except in prostate. Conclusions: P. betel leaf stalk extract exert its anti androgenic effect by alterations in rate of protein synthesis and cellular hypertrophy occur in prostate.

  4. Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants.

    Science.gov (United States)

    Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; Nguyen, Thuy N; Gidda, Satinder K; Watt, Samantha C; Yurchenko, Olga; Park, Sunjung; Sturtevant, Drew; Mullen, Robert T; Dyer, John M; Chapman, Kent D

    2017-07-01

    Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Evaluation of heat shock protein (HSP-60) induction on accumulation of carbohydrate in Isochrysis galbana

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, H.; Wolfe, M.; Tell, J.; Tjeerdema, R. [Univ. of California, Santa Cruz, CA (United States). Dept. of Chemistry and Biochemistry

    1995-12-31

    Primary levels of the marine food chain may play an important role in the fate of petroleum hydrocarbons in both chemically dispersed and un-dispersed oil spills. HSP-60 proteins, members of the chaperonin family of stress proteins, are induced in response to a wide variety of environmental agents, including UV light, heavy metals, and xenobiotics. Increased production and storage of carbohydrate in I. galbana has been associated with aging and stress. Thus, HSP-60 and carbohydrate storage were selected as sublethal endpoints of exposure to the primary producer, I. galbana, a golden brown, unicellular algae, and a significant component of the marine phytoplankton community. The authors have found that I. galbana cultures exposed to water-accommodated fractions (WAF) of Prudhoe Bay Crude Oil (PBCO), and PBCO/dispersant preparations efficiently induce HSP-60. Studies indicated that WAF produced a dose-related response in I. galbana, which increased as a function of time. Dispersant alone showed the greatest induction, while combined WAF-dispersant showed less induction, suggesting a possible competition between crude oil and algae for dispersant interaction. In addition, they have demonstrated that I. galbana accumulates carbohydrates in response to exposure to WAF and PBCO/dispersant preparations and therefore represents another index of stress in this organism. They were interested in determining if induction of stress proteins and HSP60 in particular represented an adaptive-mechanism, allowing this algae to better cope with exposure to petroleum hydrocarbons released in the marine environment during an oil spill. In an effort to determine if stress protein induction serves as a protective adaptive response to exposure to petroleum hydrocarbons they examined the effect of heat shock induction on the accumulation of carbohydrates by these organisms in response to exposure to WAF and dispersed oil preparations.

  6. HMGB1 and RAGE in skeletal muscle inflammation: Implications for protein accumulation in inclusion body myositis.

    Science.gov (United States)

    Muth, Ingrid E; Zschüntzsch, Jana; Kleinschnitz, Konstanze; Wrede, Arne; Gerhardt, Ellen; Balcarek, Peter; Schreiber-Katz, Olivia; Zierz, Stephan; Dalakas, Marinos C; Voll, Reinhard E; Schmidt, Jens

    2015-09-01

    Inflammation is associated with protein accumulation in IBM, but precise mechanisms are elusive. The "alarmin" HMGB1 is upregulated in muscle inflammation. Its receptor RAGE is crucial for β-amyloid-associated neurodegeneration. Relevant signaling via HMGB1/RAGE is expected in IBM pathology. By real-time-PCR, mRNA-expression levels of HMGB1 and RAGE were upregulated in muscle biopsies of patients with IBM and PM, but not in muscular dystrophy or non-myopathic controls. By immunohistochemistry, both molecules displayed the highest signal in IBM, where they distinctly co-localized to intra-fiber accumulations of β-amyloid and neurofilament/tau. In these fibers, identification of phosphorylated Erk suggested that relevant downstream activation is present upon HMGB1 signaling via RAGE. Protein expressions of HMGB1, RAGE, Erk and phosphorylated Erk were confirmed by Western blot. In a well established cell-culture model for pro-inflammatory cell-stress, exposure of human muscle-cells to IL-1β+IFN-γ induced cytoplasmic translocation of HMGB1 and subsequent release as evidenced by ELISA. Upregulation of RAGE on the cell surface was demonstrated by immunocytochemistry and flow-cytometry. Recombinant HMGB1 was equally potent as IL-1β+IFN-γ in causing amyloid-accumulation and cell-death, and both were abrogated by the HMGB1-blocker BoxA. The findings strengthen the concept of unique interactions between degenerative and inflammatory mechanisms and suggest that HMGB1/RAGE signaling is a critical pathway in IBM pathology.

  7. A Cooperative Approach for the Extraction of Protein Motifs

    Institute of Scientific and Technical Information of China (English)

    Chao CHEN; Yuan Xin TIAN; Xiao Yong ZOU; Pei Xiang CAI; Jin Yuan MO

    2006-01-01

    By integrating the concept of cooperative approach, an extension of the fast annealing coevolutionary algorithm is presented in this paper. It outperformed the original algorithm in the domain of function optimization, especially in terms of convergence rate. It was also applied to a real optimization problem, protein motif extraction. And a satisfactory result has been obtained with the accuracy of prediction achieving 67.0%, which is in agreement with the result in the PROSITE database.

  8. Automatically extracting functionally equivalent proteins from SwissProt

    Directory of Open Access Journals (Sweden)

    Martin Andrew CR

    2008-10-01

    Full Text Available Abstract Background There is a frequent need to obtain sets of functionally equivalent homologous proteins (FEPs from different species. While it is usually the case that orthology implies functional equivalence, this is not always true; therefore datasets of orthologous proteins are not appropriate. The information relevant to extracting FEPs is contained in databanks such as UniProtKB/Swiss-Prot and a manual analysis of these data allow FEPs to be extracted on a one-off basis. However there has been no resource allowing the easy, automatic extraction of groups of FEPs – for example, all instances of protein C. We have developed FOSTA, an automatically generated database of FEPs annotated as having the same function in UniProtKB/Swiss-Prot which can be used for large-scale analysis. The method builds a candidate list of homologues and filters out functionally diverged proteins on the basis of functional annotations using a simple text mining approach. Results Large scale evaluation of our FEP extraction method is difficult as there is no gold-standard dataset against which the method can be benchmarked. However, a manual analysis of five protein families confirmed a high level of performance. A more extensive comparison with two manually verified functional equivalence datasets also demonstrated very good performance. Conclusion In summary, FOSTA provides an automated analysis of annotations in UniProtKB/Swiss-Prot to enable groups of proteins already annotated as functionally equivalent, to be extracted. Our results demonstrate that the vast majority of UniProtKB/Swiss-Prot functional annotations are of high quality, and that FOSTA can interpret annotations successfully. Where FOSTA is not successful, we are able to highlight inconsistencies in UniProtKB/Swiss-Prot annotation. Most of these would have presented equal difficulties for manual interpretation of annotations. We discuss limitations and possible future extensions to FOSTA, and

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

    Science.gov (United States)

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

    2015-11-01

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

  10. Accumulation of C-reactive protein in basal keratinocytes of normal skins.

    Science.gov (United States)

    Ono, Koji; Fujimoto, Norihiro; Akiyama, Minoru; Satoh, Takahiro; Tajima, Shingo

    2016-07-01

    C-reactive protein (CRP) is a prototypic acute phase protein which increases dramatically in the blood during the first 48h of tissue inflammation and has been recognized as a risk factor for atherosclerosis. CRP interacts with a variety of proteins. To know the role of accumulated CRP in the skin. Interaction of CRP with basal keratinocytes was studied using immunohistochemical method and keratinocyte culture system. We found an immunohistochemical deposition of CRP on the basal keratinocyte membrane in some normal human skins (23 out of 46 skins). When added to cultured keratinocytes, heat-denatured but not native CRP was found to adhere to keratinocyte cell membrane after 1h, then internalized into cytoplasm after 24h. The heat-denatured CRP recognized at least four keratinocyte polypeptides with the molecular weights of 56, 42, 32 and 24kDa. Ligand binding assays suggested that multiple populations of receptor-ligand interactions were involved in the binding between CRP and keratinocyte. Cultured dermal microvascular endothelial cells were found to express CRP of which expression was greatly induced by interleukin-1β (IL-1β) treatment, suggesting that the deposited CRP in the basal keratinocytes can be derived from local dermal microvasculatures as well as from systemic circulation (serum). Treatment of cultured keratinocytes with heat-denatured CRP induced interleukin-8 (IL-8) expression, a potent leukocyte chemotactic cytokine. CRP in the medium (liquid phase) and CRP-coated dishes (solid phase) both inhibited the adhesion of keratinocytes in culture. Accumulation of CRP may regulate the skin inflammation and keratinocyte proliferation by modulating keratinocyte cytokine expression and adhesion to substrate. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  11. RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

    Science.gov (United States)

    Jia, Daile; He, Yuhu; Zhu, Qian; Liu, Huan; Zuo, Caojian; Chen, Guilin; Yu, Ying; Lu, Ankang

    2017-05-01

    Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for

  12. Extraction and characterisation of protein fractions from five insect species.

    Science.gov (United States)

    Yi, Liya; Lakemond, Catriona M M; Sagis, Leonard M C; Eisner-Schadler, Verena; van Huis, Arnold; van Boekel, Martinus A J S

    2013-12-15

    Tenebrio molitor, Zophobas morio, Alphitobius diaperinus, Acheta domesticus and Blaptica dubia were evaluated for their potential as a future protein source. Crude protein content ranged from 19% to 22% (Dumas analysis). Essential amino acid levels in all insect species were comparable with soybean proteins, but lower than for casein. After aqueous extraction, next to a fat fraction, a supernatant, pellet, and residue were obtained, containing 17-23%, 33-39%, 31-47% of total protein, respectively. At 3% (w/v), supernatant fractions did not form stable foams and gels at pH 3, 5, 7, and 10, except for gelation for A. domesticus at pH 7. At 30% w/v, gels at pH 7 and pH 10 were formed, but not at pH 3 and pH 5. In conclusion, the insect species studied have potential to be used in foods due to: (1) absolute protein levels; (2) protein quality; (3) ability to form gels.

  13. Evaluation of the arsenic binding capacity of plant proteins under conditions of protein extraction for gel electrophoretic analysis.

    Science.gov (United States)

    Schmidt, Anne-Christine; Steier, Sandra; Otto, Matthias

    2009-03-15

    As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80+/-8% extraction yield obtained without SDS to 48+/-2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4+/-0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 microgmL(-1) depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kgproteinmass)(-1) resulted for spiking concentrations of 10 mgAsL(-1). The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.

  14. MYC2, MYC3, and MYC4 function redundantly in seed storage protein accumulation in Arabidopsis.

    Science.gov (United States)

    Gao, Chenhao; Qi, Shuanghui; Liu, Kaige; Li, Dong; Jin, Changyu; Li, Zhuowei; Huang, Gengqing; Hai, Jiangbo; Zhang, Meng; Chen, Mingxun

    2016-11-01

    Basic helix-loop-helix transcription factors (TFs), namely MYC2, MYC3, and MYC4, interact with Jasmonate Zim-domain proteins and are their direct targets. These TFs have been shown to function synergistically to control Arabidopsis growth and development. Our results showed similar MYC2, MYC3, and MYC4 expression patterns during Arabidopsis seed development, which remained relatively high during seed mid-maturation. MYC2, MYC3, and MYC4 acted redundantly in seed size, weight control, and in regulating seed storage protein accumulation. Triple mutants produced the largest seeds and single and double mutants' seeds were much larger than those of wild type. The weight of triple mutants' seeds was significantly higher than that of wild-type seeds, which was accompanied by an increase in seed storage protein contents. Triple mutants' seeds presented a marked decrease in 2S amounts relative to those in wild-type seeds. Liquid chromatography tandem mass spectra sequencing results indicated that both the relative abundance and the peptide number of CRA1 and CRU3 were greatly increased in triple mutants compared to wild type. The expression of 2S1-2S5 decreased and that of CRA1 and CRU3 increased in triple mutants relative to those in wild types during seed development, which might have contributed to the low 2S and high 12S contents in triple mutants. Our results contribute to understanding the function of MYC2, MYC3, and MYC4 on seed development, and provide promising targets for genetic manipulations of protein-producing crops to improve the quantity and quality of seed storage proteins. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Characterization of in vivo phosphorylation modification of differentially accumulated proteins in cotton fiber-initiation process.

    Science.gov (United States)

    Liu, Wenying; Zhang, Bing; He, Wenying; Wang, Zi; Li, Guanqiao; Liu, Jinyuan

    2016-08-01

    Initiation of cotton fiber from ovule epidermal cells determines the ultimate number of fibers per cotton ovule, making it one of the restriction factors of cotton fiber yield. Previous comparative proteomics studies have collectively revealed 162 important differentially accumulated proteins (DAPs) in cotton fiber-initiation process, however, whether and how post-translational modifications, especially phosphorylation modification, regulate the expression and function of the DAPs are still unclear. Here we reported the successful identification of 17 phosphopeptides from 16 phosphoproteins out of the 162 DAPs using the integrated bioinformatics analyses of peptide mass fingerprinting data and targeted MS/MS identification method. In-depth analyses indicated that 15 of the 17 phosphorylation sites were novel phosphorylation sites first identified in plants, whereas 6 of the 16 phosphoproteins were found to be the phosphorylated isoforms of 6 proteins. The phosphorylation-regulated dynamic protein network derived from this study not only expanded our understanding of the cotton fiber-initiation process, but also provided a valuable resource for future functional studies of the phosphoproteins. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.

    Science.gov (United States)

    Menassa, Rima; Zhu, Hong; Karatzas, Costas N; Lazaris, Anthoula; Richman, Alex; Brandle, Jim

    2004-09-01

    Spider dragline silk is a unique biomaterial and represents nature's strongest known fibre. As it is almost as strong as many commercial synthetic fibres, it is suitable for use in many industrial and medical applications. The prerequisite for such a widespread use is the cost-effective production in sufficient quantities for commercial fibre manufacturing. Agricultural biotechnology and the production of recombinant dragline silk proteins in transgenic plants offer the potential for low-cost, large-scale production. The purpose of this work was to examine the feasibility of producing the two protein components of dragline silk (MaSp1 and MaSp2) from Nephila clavipes in transgenic tobacco. Two different promoters, the enhanced CaMV 35S promoter (Kay et al., 1987) and a new tobacco cryptic constitutive promoter, tCUP (Foster et al., 1999) were used, in conjunction with a plant secretory signal (PR1b), a translational enhancer (alfalfa mosaic virus, AMV) and an endoplasmic reticulum (ER) retention signal (KDEL), to express the MaSp1 and MaSp2 genes in the leaves of transgenic plants. Both genes expressed successfully and recombinant protein accumulated in transgenic plants grown in both greenhouse and field trials.

  17. Pathogenesis-related protein expression in the apoplast of wheat leaves protected against leaf rust following application of plant extracts.

    Science.gov (United States)

    Naz, Rabia; Bano, Asghari; Wilson, Neil L; Guest, David; Roberts, Thomas H

    2014-09-01

    Leaf rust (Puccinia triticina) is a major disease of wheat. We tested aqueous leaf extracts of Jacaranda mimosifolia (Bignoniaceae), Thevetia peruviana (Apocynaceae), and Calotropis procera (Apocynaceae) for their ability to protect wheat from leaf rust. Extracts from all three species inhibited P. triticina urediniospore germination in vitro. Plants sprayed with extracts before inoculation developed significantly lower levels of disease incidence (number of plants infected) than unsprayed, inoculated controls. Sprays combining 0.6% leaf extracts and 2 mM salicylic acid with the fungicide Amistar Xtra at 0.05% (azoxystrobin at 10 μg/liter + cyproconazole at 4 μg/liter) reduced disease incidence significantly more effectively than sprays of fungicide at 0.1% alone. Extracts of J. mimosifolia were most active, either alone (1.2%) or in lower doses (0.6%) in combination with 0.05% Amistar Xtra. Leaf extracts combined with fungicide strongly stimulated defense-related gene expression and the subsequent accumulation of pathogenesis-related (PR) proteins in the apoplast of inoculated wheat leaves. The level of protection afforded was significantly correlated with the ability of extracts to increase PR protein expression. We conclude that pretreatment of wheat leaves with spray formulations containing previously untested plant leaf extracts enhances protection against leaf rust provided by fungicide sprays, offering an alternative disease management strategy.

  18. LDL Receptor-Related Protein-1 (LRP1 Regulates Cholesterol Accumulation in Macrophages.

    Directory of Open Access Journals (Sweden)

    Anna P Lillis

    Full Text Available Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1 to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR-deficient background (macLRP1-/-. After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.

  19. Effect of pH and postmortem aging on protein extraction from broiler breast muscle.

    Science.gov (United States)

    Eady, M; Samuel, D; Bowker, B

    2014-07-01

    This study determined the effects of extraction buffer pH and postmortem aging on the extraction of salt-soluble and water-soluble proteins from broiler pectoralis muscle. Deboned broiler breast fillets were collected at 4 h postmortem, packaged, and then stored at 4°C until 1, 5, or 8 d postmortem. After the designated aging period, salt-soluble and water-soluble protein extractions were performed using buffers at 7 different pH levels (pH 5.4, 6.4, 6.9, 7.2, 7.5, 8.0, 9.0). Protein concentrations of the extracts were measured and SDS-PAGE analysis was performed. Salt-soluble protein concentration increased (P protein concentration increased (P extraction buffer pH by aging treatment interaction for the total protein concentration of either the salt-soluble or water-soluble protein extracts. The protein concentrations of salt-soluble extracts were similar at both 1 and 8 d postmortem but lower (P protein concentrations of water-soluble extracts were similar at both 1 and 5 d postmortem, but higher (P extraction buffer pH and postmortem aging influenced the SDS-PAGE protein profiles of salt-soluble and water-soluble protein extracts from breast muscles. Data demonstrate that postmortem aging and extraction buffer pH influence both the total amount and the composition of the myofibrillar and sarcoplasmic proteins that can be extracted from broiler breast fillets.

  20. Protein phosphorylation is involved in the water stress induced ABA accumulation in the roots of Malus hupehensis Rehd

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Water stress-induced ABA accumulation plays a key role in the root to shoot communication and/or the cell to cell signaling under the soil stresses. The signaling of the water stress itself that leads to the accumulation, however, is less known. In this study, we subjected the roots of Malus hupehensis seedlings to water stress treatment and investigated the ABA accumulation in relation to protein phosphorylation. Our results showed that ABA accumulation could be substantially triggered in 40 min and reached 4 folds in 100 min after treatment with 30% PEG 6000 (polyethylene glycol). The water stress treatment also led to a substantial enhancement of total kinase activity, assessed with histone-Ⅲ as substrate, in 15 min and a maximum enhancement in 30 min before it declined to initial level. The Ca2+-dependent kinase activity showed a similar, if not more sensitive, trend. When the roots were fed with labeled 32pATP, water stress enhanced the labeling of proteins, which showed a maximum labeling at 40 min. Two inhibitors of protein kinases, Quercetin and H7, effectively diminished or completely blocked the ABA accumulation under the stress treatment. It is therefore suggest that protein phosphorylation is involved in the signaling of the water stress-induced ABA accumulation.

  1. Chronic Exposure to Rhodobacter Sphaeroides Extract Lycogen™ Prevents UVA-Induced Malondialdehyde Accumulation and Procollagen I Down-Regulation in Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Tsai-Hsiu Yang

    2014-01-01

    Full Text Available UVA contributes to the pathogenesis of skin aging by downregulation of procollagen I content and induction of matrix metalloproteinase (MMP-associated responses. Application of antioxidants such as lycopene has been demonstrated as a convenient way to achieve protection against skin aging. Lycogen™, derived from the extracts of Rhodobacter sphaeroides, exerts several biological effects similar to that of lycopene whereas most of its anti-aging efficacy remains uncertain. In this study, we attempted to examine whether Lycogen™ could suppress malondialdehyde (MDA accumulation and restore downregulated procollagen I expression induced by UVA exposure. In human dermal fibroblasts Hs68 cells, UVA repressed cell viability and decreased procollagen I protein content accompanied with the induction of MMP-1 and MDA accumulation. Remarkably, incubation with 50 µM Lycogen™ for 24 h ameliorated UVA-induced cell death and restored UVA-induced downregulation of procollagen in a dose-related manner. Lycogen™ treatment also prevented the UVA-induced MMP-1 upregulation and intracellular MDA generation in Hs68 cells. Activation of NFκB levels, one of the downstream events induced by UVA irradiation and MMP-1 induction, were also prevented by Lycogen™ administration. Taken together, our findings demonstrate that Lycogen™ may be an alternative agent that prevents UVA-induced skin aging and could be used in cosmetic and pharmaceutical applications.

  2. Soy Leaf Extract Containing Kaempferol Glycosides and Pheophorbides Improves Glucose Homeostasis by Enhancing Pancreatic β-Cell Function and Suppressing Hepatic Lipid Accumulation in db/db Mice.

    Science.gov (United States)

    Li, Hua; Ji, Hyeon-Seon; Kang, Ji-Hyun; Shin, Dong-Ha; Park, Ho-Yong; Choi, Myung-Sook; Lee, Chul-Ho; Lee, In-Kyung; Yun, Bong-Sik; Jeong, Tae-Sook

    2015-08-19

    This study investigated the molecular mechanisms underlying the antidiabetic effect of an ethanol extract of soy leaves (ESL) in db/db mice. Control groups (db/+ and db/db) were fed a normal diet (ND), whereas the db/db-ESL group was fed ND with 1% ESL for 8 weeks. Dietary ESL improved glucose tolerance and lowered plasma glucose, glycated hemoglobin, HOMA-IR, and triglyceride levels. The pancreatic insulin content of the db/db-ESL group was significantly greater than that of the db/db group. ESL supplementation altered pancreatic IRS1, IRS2, Pdx1, Ngn3, Pax4, Ins1, Ins2, and FoxO1 expression. Furthermore, ESL suppressed lipid accumulation and increased glucokinase activity in the liver. ESL primarily contained kaempferol glycosides and pheophorbides. Kaempferol, an aglycone of kaempferol glycosides, improved β-cell proliferation through IRS2-related FoxO1 signaling, whereas pheophorbide a, a product of chlorophyll breakdown, improved insulin secretion and β-cell proliferation through IRS1-related signaling with protein kinase A in MIN6 cells. ESL effectively regulates glucose homeostasis by enhancing IRS-mediated β-cell insulin signaling and suppressing SREBP-1-mediated hepatic lipid accumulation in db/db mice.

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

    Science.gov (United States)

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

    2017-02-15

    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.

  4. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms.

    Directory of Open Access Journals (Sweden)

    Pedro Fernandez-Funez

    2009-06-01

    Full Text Available Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C converts into a misfolded isoform (PrP(Sc with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc-specific conformational epitopes. In contrast to PrP(Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP(Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover

  5. Cadmium and manganese accumulation in Phytolacca americana L. and the roles of non-protein thiols and organic acids.

    Science.gov (United States)

    Gao, Lu; Peng, Kejian; Xia, Yan; Wang, Guiping; Niu, Liyuan; Lian, Chunlan; Shen, Zhenguo

    2013-01-01

    Phytolacca americana L. can accumulate large amounts of heavy metals in its aerial tissues, especially cadmium (Cd) and manganese (Mn). It has great potential for use in phytoextraction of metals from multi-metal-contaminated soils. This study was conducted to further investigate the Cd- and Mn-tolerance strategies of this plant. Concentrations of non-protein thiols (NPTs) and phytochelatins (PCs) in leaves and roots increased significantly as the concentration of Cd in solution increased. The molar ratios of PCs:soluble Cd ranged from 1.8 to 3.6 in roots and 8.1 to 31.6 in leaves, suggesting that the cellular response involving PC synthesis was sufficient to complex Cd ions in the cytosol, especially that of leaves. In contrast, excess Mn treatments did not result in a significant increase in NPT or PC concentrations in leaves or roots. Oxalic acid concentrations in leaves of plants exposed to 2 or 20 mM Mn reached 69.4 to 89.3 mg (0.771 to 0.992 mmol) g(-1) dry weight, respectively, which was approximately 3.7- to 8.6-fold higher than the Mn level in the 0.6 M HCl extract. Thus, oxalic acid may play an important role in the detoxification of Mn.

  6. Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

    OpenAIRE

    2016-01-01

    Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as you...

  7. An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

    Science.gov (United States)

    Tan, Niu J; Daim, Leona D J; Jamil, Amilia A M; Mohtarrudin, Norhafizah; Thilakavathy, Karuppiah

    2017-03-01

    Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Tree kernel-based protein-protein interaction extraction from biomedical literature.

    Science.gov (United States)

    Qian, Longhua; Zhou, Guodong

    2012-06-01

    There is a surge of research interest in protein-protein interaction (PPI) extraction from biomedical literature. While most of the state-of-the-art PPI extraction systems focus on dependency-based structured information, the rich structured information inherent in constituent parse trees has not been extensively explored for PPI extraction. In this paper, we propose a novel approach to tree kernel-based PPI extraction, where the tree representation generated from a constituent syntactic parser is further refined using the shortest dependency path between two proteins derived from a dependency parser. Specifically, all the constituent tree nodes associated with the nodes on the shortest dependency path are kept intact, while other nodes are removed safely to make the constituent tree concise and precise for PPI extraction. Compared with previously used constituent tree setups, our dependency-motivated constituent tree setup achieves the best results across five commonly used PPI corpora. Moreover, our tree kernel-based method outperforms other single kernel-based ones and performs comparably with some multiple kernel ones on the most commonly tested AIMed corpus.

  9. Hibiscus sabdariffa extract inhibits obesity and fat accumulation, and improves liver steatosis in humans.

    Science.gov (United States)

    Chang, Hong-Chou; Peng, Chiung-Huei; Yeh, Da-Ming; Kao, Erl-Shyh; Wang, Chau-Jong

    2014-04-01

    Obesity is associated with a great diversity of diseases including non-alcoholic fatty liver disease. Our previous report suggested that Hibiscus sabdariffa extracts (HSE) had a metabolic-regulating and liver-protecting potential. In this study, we performed a clinical trial to further confirm the effect of HSE. Subjects with a BMI ≧ 27 and aged 18-65, were randomly divided into control (n = 17) and HSE-treated (n = 19) groups, respectively, for 12 weeks. Our data showed that consumption of HSE reduced body weight, BMI, body fat and the waist-to-hip ratio. Serum free fatty acid (FFA) was lowered by HSE. Anatomic changes revealed that HSE improved the illness of liver steatosis. Ingestion of HSE was well tolerated and there was no adverse effect during the trial. No alteration was found for serum α-amylase and lipase. The clinical effect should mainly be attributed to the polyphenols of HSE, since composition analysis showed that branched chain-amino acids, which is associated with obesity, is not obviously high. In conclusion, consumption of HSE reduced obesity, abdominal fat, serum FFA and improved liver steatosis. HSE could act as an adjuvant for preventing obesity and non-alcoholic fatty liver.

  10. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma.

    Science.gov (United States)

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-10-15

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients.

  11. Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis.

    Science.gov (United States)

    Fowler, Carol B; Waybright, Timothy J; Veenstra, Timothy D; O'Leary, Timothy J; Mason, Jeffrey T

    2012-04-06

    Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.

  12. Integrating semantic information into multiple kernels for protein-protein interaction extraction from biomedical literatures.

    Directory of Open Access Journals (Sweden)

    Lishuang Li

    Full Text Available Protein-Protein Interaction (PPI extraction is an important task in the biomedical information extraction. Presently, many machine learning methods for PPI extraction have achieved promising results. However, the performance is still not satisfactory. One reason is that the semantic resources were basically ignored. In this paper, we propose a multiple-kernel learning-based approach to extract PPIs, combining the feature-based kernel, tree kernel and semantic kernel. Particularly, we extend the shortest path-enclosed tree kernel (SPT by a dynamic extended strategy to retrieve the richer syntactic information. Our semantic kernel calculates the protein-protein pair similarity and the context similarity based on two semantic resources: WordNet and Medical Subject Heading (MeSH. We evaluate our method with Support Vector Machine (SVM and achieve an F-score of 69.40% and an AUC of 92.00%, which show that our method outperforms most of the state-of-the-art systems by integrating semantic information.

  13. Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Krochko, J E; Pramanik, S K; Bewley, J D

    1992-05-01

    During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media

  14. A Shortest Dependency Path Based Convolutional Neural Network for Protein-Protein Relation Extraction

    Directory of Open Access Journals (Sweden)

    Lei Hua

    2016-01-01

    Full Text Available The state-of-the-art methods for protein-protein interaction (PPI extraction are primarily based on kernel methods, and their performances strongly depend on the handcraft features. In this paper, we tackle PPI extraction by using convolutional neural networks (CNN and propose a shortest dependency path based CNN (sdpCNN model. The proposed method (1 only takes the sdp and word embedding as input and (2 could avoid bias from feature selection by using CNN. We performed experiments on standard Aimed and BioInfer datasets, and the experimental results demonstrated that our approach outperformed state-of-the-art kernel based methods. In particular, by tracking the sdpCNN model, we find that sdpCNN could extract key features automatically and it is verified that pretrained word embedding is crucial in PPI task.

  15. A Shortest Dependency Path Based Convolutional Neural Network for Protein-Protein Relation Extraction.

    Science.gov (United States)

    Hua, Lei; Quan, Chanqin

    2016-01-01

    The state-of-the-art methods for protein-protein interaction (PPI) extraction are primarily based on kernel methods, and their performances strongly depend on the handcraft features. In this paper, we tackle PPI extraction by using convolutional neural networks (CNN) and propose a shortest dependency path based CNN (sdpCNN) model. The proposed method (1) only takes the sdp and word embedding as input and (2) could avoid bias from feature selection by using CNN. We performed experiments on standard Aimed and BioInfer datasets, and the experimental results demonstrated that our approach outperformed state-of-the-art kernel based methods. In particular, by tracking the sdpCNN model, we find that sdpCNN could extract key features automatically and it is verified that pretrained word embedding is crucial in PPI task.

  16. Fenugreek Seed Extract Inhibit Fat Accumulation and Ameliorates Dyslipidemia in High Fat Diet-Induced Obese Rats

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    2014-01-01

    Full Text Available This study investigated the inhibitory effect of aqueous extract of Trigonella foenum-graecum seeds (AqE-TFG on fat accumulation and dyslipidemia in high fat diet- (HFD- induced obese rats. Female Wistar rats were fed with HFD ad libitum, and the rats on HFD were treated orally with AqE-TFG or orlistat ((HFD for 28 days + AqE-TFG (0.5 and 1.0 g/kg or orlistat (10 mg/kg from day 8 to 28, respectively. Treatment with AqE-TFG produced significant reduction in body weight gain, body mass index (BMI, white adipose tissue (WAT weights, blood glucose, serum insulin, lipids, leptin, lipase, and apolipoprotein-B levels and elevation in adiponectin levels. AqE-TFG improved serum aspartate amino transferase (AST, alanine amino transferase (ALT, and lactate dehydrogenase (LDH levels. AqE-TFG treatment reduced the hepatic and cardiac thiobarbituric acid reactive substances (TBARS and elevated the antioxidant enzyme (glutathione (GSH, superoxide dismutase (SOD, and catalase (CAT levels. In addition, liver and uterine WAT lipogenic enzyme (fatty acid synthetase (FAS and glucose-6-phosphate dehydrogenase (G6PD activities were restored towards normal levels. These findings demonstrated the preventive effect of AqE-TFG on fat accumulation and dyslipidemia, due to inhibition of impaired lipid digestion and absorption, in addition to improvement in glucose and lipid metabolism, enhancement of insulin sensitivity, increased antioxidant defense, and downregulation of lipogenic enzymes.

  17. Formation of the Yandangshan volcanic-plutonic complex (SE China) by melt extraction and crystal accumulation

    Science.gov (United States)

    Yan, Li-Li; He, Zhen-Yu; Jahn, Bor-ming; Zhao, Zhi-Dan

    2016-12-01

    The association of volcanic and shallow plutonic rocks in caldera may provide important clues to the geochemical evolution of silicic magma systems. The Yandangshan caldera is a typical example of late Mesozoic volcanic-plutonic complex in SE China. It is composed of a series of rhyolitic extrusives and subvolcanic intrusions of porphyritic quartz syenites. In this work, we conducted petrological and geochemical studies, as well as zircon dating, on the coexisting volcanic and plutonic rocks from the Yandangshan caldera. The results of SHRIMP and LA-ICP-MS zircon U-Pb dating revealed that the crystallization of the rhyolitic extrusives and subvolcanic intrusions was contemporaneous within analytical errors and in a short period (104-98 Ma). Geochemically, the volcanic rocks are characterized by high Rb/Sr and Rb/Ba ratios and depletion in Ba, Sr, P, Eu and Ti, while the shallow plutons show high K, Ba, Al, Fe and low Rb/Sr and Rb/Ba ratios with insignificant negative Eu anomalies. The volcanic and plutonic rocks have a similar range of zircon Hf isotopic compositions (εHf(t) = - 10.0 to + 1.5) and TDM2 model ages of 2.10-1.23 Ga. They also have comparable whole-rock Sr and Nd isotopic compositions ((87Sr/86Sr)i = 0.7084-0.7090; εNd(t) = - 7.8 to - 6.5) and zircon oxygen isotopic compositions (δ18O mainly = 4.5 to 6.0‰). We argue that the volcanic-plutonic complex of the Yandangshan caldera was formed by reworking of Paleoproterozoic lower crusts in the eastern Cathaysia block, and that the complex could be linked by fractional crystallization and crystal accumulation in a shallow magma chamber. The volcanic rocks represent the highly fractionated end-member, whereas the subvolcanic intrusions of porphyritic quartz syenites could be the residual crystal mushes. This case study could have a general implication for the genetic relationship between volcanic and shallow plutonic rocks in calderas.

  18. Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

    Science.gov (United States)

    Luís, Inês M.; Alexandre, Bruno M.; Oliveira, M. Margarida

    2016-01-01

    Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK. PMID

  19. Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer brain and the possible mechanism

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    SMI34 were increased, and the elevated p-NF-H/M tended to be condensed in the proximal end of the cell processes after treated with 15 nmol/L OA. Further accumulation of p-NF-H/M to the cell plasma and parikarya was seen after increasing the concentration of OA to 30 nmol/L. On the other hand, the majority of np-NF-H/M bound to SMI32 and SMI33 were seen in the cell body although it was also detected in cell processes before OA treatment. The immunoreaction of np-NF-H/M was significantly decreased in the cell body and it became to be condensed in the proximal end of the cell processes after treatment of the cell by 15 nmol/L of OA. Further decreasing of the staining was observed when the concentration of OA was raised to 30 nmol/L. The data demonstrated that an Alzheimer-like inhibition of PP-2A and PP-1 induced hyperphosphorylation and accumulation of NF proteins as seen in AD brain, indicating that abnormality of NF might be involved in AD neurofibrillary degeneration. As SY5Y contains negligible amount of tau protein which was reported to cross-react with p-NF subunits, it might be served as a proper cell model for NF study.

  20. Mango (Mangifera indica L.) peel extract fractions from different cultivars differentially affect lipid accumulation in 3T3-L1 adipocyte cells.

    Science.gov (United States)

    Taing, Meng-Wong; Pierson, Jean-Thomas; Shaw, Paul N; Dietzgen, Ralf G; Roberts-Thomson, Sarah J; Gidley, Michael J; Monteith, Gregory R

    2013-02-26

    Plant phytochemicals are increasingly recognised as sources of bioactive molecules which may have potential benefit in many health conditions. In mangoes, peel extracts from different cultivars exhibit varying effects on adipogenesis in the 3T3-L1 adipocyte cell line. In this study, the effects of preparative HPLC fractions of methanol peel extracts from Irwin, Nam Doc Mai and Kensington Pride mangoes were evaluated. Fraction 1 contained the most hydrophilic components while subsequent fractions contained increasingly more hydrophobic components. High content imaging was used to assess mango peel fraction effects on lipid accumulation, nuclei count and nuclear area in differentiating 3T3-L1 cells. For all three mango cultivars, the more hydrophilic peel fractions 1-3 inhibited lipid accumulation with greater potency than the more hydrophobic peel fractions 4. For all three cultivars, the more lipophilic fraction 4 had concentrations that enhanced lipid accumulation greater than fractions 1-3 as assessed by lipid droplet integrated intensity. The potency of this fraction 4 varied significantly between cultivars. Using mass spectrometry, five long chain free fatty acids were detected in fraction 4; these were not present in any other peel extract fractions. Total levels varied between cultivars, with Irwin fraction 4 containing the highest levels of these free fatty acids. Lipophilic components appear to be responsible for the lipid accumulation promoting effects of some mango extracts and are the likely cause of the diverse effects of peel extracts from different mango cultivars on lipid accumulation.

  1. DNA, RNA, and Protein Extraction: The Past and The Present

    Directory of Open Access Journals (Sweden)

    Beow Chin Yiap

    2009-01-01

    Full Text Available Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.

  2. Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host

    Directory of Open Access Journals (Sweden)

    Warner Richard

    2009-02-01

    Full Text Available Abstract Background In order to study the sites of uptake and mechanisms of dissemination of scrapie prions in the natural host under controlled conditions, lambs aged 14 days and homozygous for the VRQ allele of the PrP gene were infected by the oral route. Infection occurred in all lambs with a remarkably short and highly consistent incubation period of approximately 6 months. Challenge of lambs at approximately eight months of age resulted in disease in all animals, but with more variable incubation periods averaging significantly longer than those challenged at 14 days. This model provides an excellent system in which to study the disease in the natural host by virtue of the relatively short incubation period and close resemblance to natural infection. Results Multiple sites of prion uptake were identified, of which the most important was the Peyer's patch of the distal ileum. Neuroinvasion was detected initially in the enteric nervous system prior to infection of the central nervous system. At end stage disease prion accumulation was widespread throughout the entire neuraxis, but vacuolar pathology was absent in most animals that developed disease at 6–7 months of age. Conclusion Initial spread of detectable PrP was consistent with drainage in afferent lymph to dependent lymph nodes. Subsequent accumulation of prions in lymphoid tissue not associated with the gut is consistent with haematogenous spread. In addition to macrophages and follicular dendritic cells, prion containing cells consistent with afferent lymph dendritic cells were identified and are suggested as a likely vehicle for carriage of prions from initial site of uptake to the lymphoreticular system, and as potential carriers of prion protein in blood. It is apparent that spongiform change, the characteristic lesion of scrapie and other prion diseases, is not responsible for the clinical signs in sheep, but may develop in an age dependent manner.

  3. Hepcidin Suppresses Brain Iron Accumulation by Downregulating Iron Transport Proteins in Iron-Overloaded Rats.

    Science.gov (United States)

    Du, Fang; Qian, Zhong-Ming; Luo, Qianqian; Yung, Wing-Ho; Ke, Ya

    2015-08-01

    Iron accumulates progressively in the brain with age, and iron-induced oxidative stress has been considered as one of the initial causes for Alzheimer's disease (AD) and Parkinson's disease (PD). Based on the role of hepcidin in peripheral organs and its expression in the brain, we hypothesized that this peptide has a role to reduce iron in the brain and hence has the potential to prevent or delay brain iron accumulation in iron-associated neurodegenerative disorders. Here, we investigated the effects of hepcidin expression adenovirus (ad-hepcidin) and hepcidin peptide on brain iron contents, iron transport across the brain-blood barrier, iron uptake and release, and also the expression of transferrin receptor-1 (TfR1), divalent metal transporter 1 (DMT1), and ferroportin 1 (Fpn1) in cultured microvascular endothelial cells and neurons. We demonstrated that hepcidin significantly reduced brain iron in iron-overloaded rats and suppressed transport of transferrin-bound iron (Tf-Fe) from the periphery into the brain. Also, the peptide significantly inhibited expression of TfR1, DMT1, and Fpn1 as well as reduced Tf-Fe and non-transferrin-bound iron uptake and iron release in cultured microvascular endothelial cells and neurons, while downregulation of hepcidin with hepcidin siRNA retrovirus generated opposite results. We concluded that, under iron-overload, hepcidin functions to reduce iron in the brain by downregulating iron transport proteins. Upregulation of brain hepcidin by ad-hepcidin emerges as a new pharmacological treatment and prevention for iron-associated neurodegenerative disorders.

  4. Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

    Science.gov (United States)

    Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

    2016-12-02

    Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

  5. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation.

    Science.gov (United States)

    Wittkop, Linda; Schwarz, Alexandra; Cassany, Aurelia; Grün-Bernhard, Stefanie; Delaleau, Mildred; Rabe, Birgit; Cazenave, Christian; Gerlich, Wolfram; Glebe, Dieter; Kann, Michael

    2010-07-01

    Capsids of hepatitis B virus and other hepadnaviruses contain a cellular protein kinase, which phosphorylates the capsid protein. Some phosphorylation sites are shown to be essential for distinct steps of viral replication as pregenome packaging or plus strand DNA synthesis. Although different protein kinases have been reported to phosphorylate the capsid protein, varying experimental approaches do not allow direct comparison. Furthermore, the activity of a specific protein kinase has not yet been correlated to steps in the hepadnaviral life cycle. In this study we show that capsids from various sources encapsidate active protein kinase Calpha, irrespective of hepatitis B virus genotype and host cell. Treatment of a virion expressing cell line with a pseudosubstrate inhibitor showed that inhibition of protein kinase C phosphorylation did not affect genome maturation but resulted in capsid accumulation and inhibited virion release to the medium. Our results imply that different protein kinases have distinct functions within the hepadnaviral life cycle.

  6. Qushi Huayu Decoction Inhibits Hepatic Lipid Accumulation by Activating AMP-Activated Protein Kinase In Vivo and In Vitro

    Directory of Open Access Journals (Sweden)

    Qin Feng

    2013-01-01

    Full Text Available Qushi Huayu Decoction (QHD, a Chinese herbal formula, has been proven effective on alleviating nonalcoholic fatty liver disease (NAFLD in human and rats. The present study was conducted to investigate whether QHD could inhibit hepatic lipid accumulation by activating AMP-activated protein kinase (AMPK in vivo and in vitro. Nonalcoholic fatty liver (NAFL model was duplicated with high-fat diet in rats and with free fatty acid (FFA in L02 cells. In in vivo experimental condition, QHD significantly decreased the accumulation of fatty droplets in livers, lowered low-density lipoprotein cholesterol (LDL-c, alanine aminotransferase (ALT, and aspartate aminotransferase (AST levels in serum. Moreover, QHD supplementation reversed the HFD-induced decrease in the phosphorylation levels of AMPK and acetyl-CoA carboxylase (ACC and decreased hepatic nuclear protein expression of sterol regulatory element-binding protein-1 (SREBP-1 and carbohydrate-responsive element-binding protein (ChREBP in the liver. In in vitro, QHD-containing serum decreased the cellular TG content and alleviated the accumulation of fatty droplets in L02 cells. QHD supplementation reversed the FFA-induced decrease in the phosphorylation levels of AMPK and ACC and decreased the hepatic nuclear protein expression of SREBP-1 and ChREBP. Overall results suggest that QHD has significant effect on inhibiting hepatic lipid accumulation via AMPK pathway in vivo and in vitro.

  7. Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase (SPS) Activity in Two Tomato Species

    Institute of Scientific and Technical Information of China (English)

    WANG Li; CUI Na; ZHAO Xiao-cui; FAN Hai-yan; LI Tian-lai

    2014-01-01

    To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase (SPS) activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography (HPLC). The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase (AI) and neutral invertase (NI) in Micro-Tom (Solanum lycopersicum) fruit, while inSolanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.

  8. Proteolysis of chloroplast proteins is responsible for accumulation of free amino acids in dark-treated tea (Camellia sinensis) leaves.

    Science.gov (United States)

    Chen, Yiyong; Fu, Xiumin; Mei, Xin; Zhou, Ying; Cheng, Sihua; Zeng, Lanting; Dong, Fang; Yang, Ziyin

    2017-02-02

    Shade management (dark treatment) on tea (Camellia sinensis) plants is a common approach to improve free amino acids in raw materials of tea leaves. However, the reason for amino acid accumulation in dark-treated tea leaves is still unknown. In the present study, dark treatment significantly increased content of free amino acids and reduced content of soluble proteins in tea leaves. Quantitative proteomics analysis showed that most enzymes involved in biosyntheses of amino acids were down-accumulated by dark treatment. Chloroplast numbers reduced in dark-treated leaves and the content of soluble proteins reduced in the chloroplasts isolated from dark-treated leaves compared to control. These suggest that proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves. Two chloroplasts proteases, ATP-dependent Clp protease proteolytic subunit 3 and protease Do-like 2, were up-accumulated in dark-treated leaves. This study firstly elucidated the mechanism of accumulation of amino acids in dark-treated tea leaves.

  9. Enzymatic aqueous technology for simultaneous coconut protein and oil extraction

    Directory of Open Access Journals (Sweden)

    Coelho, Maria A. Z.

    2003-03-01

    Full Text Available In this study, the following commercial enzymes were evaluated in the enzymatic extraction of oil and protein from coconut: Celluclast, Termamyl, Viscozyme, Neutrase y Protease. Preliminary experiments were carried out for selection of enzymes, enzyme concentration and incubation time. In order to verify the effects of pH and water/substrate ratio, a response surface methodology was applied based in a 32 full factorial experimental design. The pH is the most meaningful parameter on oil and protein extraction yields, with a significance level higher than 90%. The maximal extraction yields of oil and protein emulsion (83% was reached using Viscozyme L and subsequently Neutrase 1.5 MG at concentrations of 0.6% (w/w and 0.3% (w/w, respectively, total incubation time of 60 minutes, substrate/water ratio 1:6 and pH around 7 (no adjustment.En este trabajo diferentes enzimas hidroliticos fueron usados para la extracción de aceite y proteína de coco: Celluclast, Termamyl, Viscozyme, Neutrase y Protease producidos por Novo Nordisk. Experimientos preliminares fueron encaminados a la selección de los extractos enzimaticos, de las concentraciones de las enzimas y de los tiempos de incubación. La técnica de análisis de varianza, mediante un delineamiento factorial en tres niveles, fue usada para la optimización de los procesos con relación a los parámetros pH y razón sustrato/agua. Comparando con la extracción acuosa todos los enzimas hidroliticos incrementan la extracción de aceite y proteína vegetal de coco. Los coeficientes de correlación muestran que el pH fué el parámetro mas significativo (pViscozyme y Neutrase 0.6 % (w/w y 0.3 % (w/w, respectivamente, tiempo de incubación 30 minutos, razón sustrato/agua 1:6 y pH 7.

  10. Exploring metal detoxification and accumulation potential during vermicomposting of Tea factory coal ash: sequential extraction and fluorescence probe analysis

    Science.gov (United States)

    Goswami, Linee; Pratihar, Sanjay; Dasgupta, Suman; Bhattacharyya, Pradip; Mudoi, Pronab; Bora, Jayanta; Bhattacharya, Satya Sundar; Kim, Ki Hyun

    2016-07-01

    Metal contamination from coal ashes (CAs) is widely recognized as a significant environmental concern. To learn more about metal detoxification and accumulation potential of earthworm species, metal-rich tea factory coal ashes (TFCA) were fed to Eisenia fetida and Lampito mauritii by employing a fluorescent tag detection method. Fascinatingly, on feeding fluorescence probed Zn and Cd along with cow dung to Eisenia fetida, the detection of the gut-proteins with a molecular mass higher than 100 kDa was a distinct evidence of metal binding. Significant increases were observed in the content of humified organic C [humic acid (HAC) and fulvic acid C (FAC)] and degree of humification during vermicomposting. Concurrently, considerably large amount of toxic metals (Cr, Cd, Pb, and Zn) was transformed from exchangeable to recalcitrant (organic matter and mineral bound) fractions. Moreover, total metal concentrations were reduced with high removal efficiency upon vermicomposting.

  11. Exploring metal detoxification and accumulation potential during vermicomposting of Tea factory coal ash: sequential extraction and fluorescence probe analysis

    Science.gov (United States)

    Goswami, Linee; Pratihar, Sanjay; Dasgupta, Suman; Bhattacharyya, Pradip; Mudoi, Pronab; Bora, Jayanta; Bhattacharya, Satya Sundar; Kim, Ki Hyun

    2016-01-01

    Metal contamination from coal ashes (CAs) is widely recognized as a significant environmental concern. To learn more about metal detoxification and accumulation potential of earthworm species, metal-rich tea factory coal ashes (TFCA) were fed to Eisenia fetida and Lampito mauritii by employing a fluorescent tag detection method. Fascinatingly, on feeding fluorescence probed Zn and Cd along with cow dung to Eisenia fetida, the detection of the gut-proteins with a molecular mass higher than 100 kDa was a distinct evidence of metal binding. Significant increases were observed in the content of humified organic C [humic acid (HAC) and fulvic acid C (FAC)] and degree of humification during vermicomposting. Concurrently, considerably large amount of toxic metals (Cr, Cd, Pb, and Zn) was transformed from exchangeable to recalcitrant (organic matter and mineral bound) fractions. Moreover, total metal concentrations were reduced with high removal efficiency upon vermicomposting. PMID:27456167

  12. Ternary diagram of extract proteins / solvent systems: Sesame, soybean and lupine proteins

    Directory of Open Access Journals (Sweden)

    Mohamed, S. S.

    2004-09-01

    Full Text Available Solvent extraction as a method of extracting protein from oilseed meals offers the advantage of higher efficiency. Unfortunately, the published literature points to the gap in the work concerned with the necessary equilibrium diagram to design due process equipment for such extracts. Initiated by this lack of basic knowledge, the present study has been undertaken to provide the equilibrium data for three different ternary systems, namely: sesame protein / sodium hydroxide solution system, soybean protein / sodium hydroxide solution system and lupine protein / sodium hydroxide solution system. These oilseed meals were selected because of their high protein content (53.4 %, 46.2 % and 42.3 % protein, respectively. The study also concentrated on the evaluation of the major parameters affecting the extraction process, i.e. the normality of the sodium hydroxide solution used as extracting solvent and the initial oilseed solvent to meal feeding ratio. The results obtained indicate that the best normality of sodium hydroxide solution used for extracting soybean and lupine protein is 0.02N, while 0.04N solution is required for extracting sesame protein. Also, operating at a liquid to solid feed ratio of 30:1 and 50:1 for soybean, sesame and lupine, respectively, is enough to reach a high protein extract. Correlations were presented for each locus of under flow compositions, graphically acquired, and the data are compared with those calculated by analytical solutions.La extracción con disolventes es un método de extracción de proteínas de las harinas de semillas oleaginosas que ofrece la ventaja de su elevada eficacia. Desafortunadamente, la bibliografía coincide en el vacío existente con respecto a los diagramas de equilibrio necesarios para el diseño de los equipos adecuados. Debido a esta falta de conocimientos, el presente estudio se ha llevado a cabo para obtener datos de tres sistemas ternarios: sistema proteína de sésamo / disolución de

  13. Phytotoxic effects of cyanobacteria extract on the aquatic plant Lemna gibba: microcystin accumulation, detoxication and oxidative stress induction.

    Science.gov (United States)

    Saqrane, Sana; Ghazali, Issam El; Ouahid, Youness; Hassni, Majida El; Hadrami, Ismaïl El; Bouarab, Lahcen; del Campo, Franscica F; Oudra, Brahim; Vasconcelos, Vitor

    2007-08-01

    The occurrence of toxic cyanobacteria in the aquatic environment constitutes a serious risk for the ecological balance and the functioning of ecosystems. The presence of cyanotoxins in ecosystems could have eventual adverse effects on aquatic plants, which play an important biological role as primary producers. The original aim of this study was to investigate microcystin (MC) accumulation, detoxication and oxidative stress induction in the free-floating aquatic vascular plant Lemna gibba (Duckweed, Lemnaceae). Experiments were carried out with a range of MC levels, obtained from toxic Microcystis culture extracts (0.075, 0.15, 0.22 and 0.3 microg equiv.MC-LR mL(-1)). During chronic exposure of the plant to MC, we examined the growth, photosynthetic pigment contents and also the physiological behavior related to toxin accumulation, possible biodegradation and stress oxidative processes of L. gibba. For the last reason, changes in peroxidase activity and phenol compound content were determined. This is a first report using phenol compounds as indicators of biotic stress induced by MC contamination in aquatic plants. Following MC exposure, a significant decrease of plant growth and chlorophyll content was observed. Also, it was demonstrated that L. gibba could take up and bio-transform microcystins. A suspected MC degradation metabolite was detected in treated Lemna cells. In response to chronic contamination with MCs, changes in the peroxidase activity and qualitative and quantitative changes in phenolic compounds were observed after 24h of plant exposure. The physiological effects induced by chronic exposure to microcystins confirm that in aquatic ecosystems plants coexisting with toxic cyanobacterial blooms may suffer an important negative ecological impact. This may represent a sanitary risk due to toxin bioaccumulation and biotransfer through the food chain.

  14. A cooperative fast annealing coevolutionary algorithm for protein motif extraction

    Institute of Scientific and Technical Information of China (English)

    CHEN Chao; TIAN YuanXin; ZOU XiaoYong; CAI PeiXiang; MO JinYuan

    2007-01-01

    By integrating the cooperative approach with the fast annealing coevolutionary algorithm (FAEA), a so-called cooperative fast annealing coevolutionary algorithm (CFACA) is presented in this paper for the purpose of solving high-dimensional problems. After the partition of the search space in CFACA, each smaller one is then searched by a separate FAEA. The fitness function is evaluated by combining sub-solutions found by each of the FAEAs. It demonstrates that the CFACA outperforms the FAEA in the domain of function optimization, especially in terms of convergence rate. The current algorithm is also applied to a real optimization problem of protein motif extraction. And a satisfactory result has been obtained with the accuracy of prediction achieving 67.0%, which is in agreement with the result in the PROSITE database.

  15. Preventing protein adsorption from a range of surfaces using an aqueous fish protein extract

    DEFF Research Database (Denmark)

    Pillai, Saju; Arpanaei, Ayyoob; Meyer, Rikke L.;

    2009-01-01

    We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO2), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed...... Fg- or HSA-coated surfaces are exposed to the FPs, a significant increase in adsorbed mass occurs because the FPs are highly surface-active displacing Fg. Additionally, fluorescence microscopy confirms that very little Fg adsorbs to the FP-coated surfaces. We propose that FP coatings prevent protein...

  16. A comprehensive benchmark of kernel methods to extract protein-protein interactions from literature.

    Science.gov (United States)

    Tikk, Domonkos; Thomas, Philippe; Palaga, Peter; Hakenberg, Jörg; Leser, Ulf

    2010-07-01

    The most important way of conveying new findings in biomedical research is scientific publication. Extraction of protein-protein interactions (PPIs) reported in scientific publications is one of the core topics of text mining in the life sciences. Recently, a new class of such methods has been proposed - convolution kernels that identify PPIs using deep parses of sentences. However, comparing published results of different PPI extraction methods is impossible due to the use of different evaluation corpora, different evaluation metrics, different tuning procedures, etc. In this paper, we study whether the reported performance metrics are robust across different corpora and learning settings and whether the use of deep parsing actually leads to an increase in extraction quality. Our ultimate goal is to identify the one method that performs best in real-life scenarios, where information extraction is performed on unseen text and not on specifically prepared evaluation data. We performed a comprehensive benchmarking of nine different methods for PPI extraction that use convolution kernels on rich linguistic information. Methods were evaluated on five different public corpora using cross-validation, cross-learning, and cross-corpus evaluation. Our study confirms that kernels using dependency trees generally outperform kernels based on syntax trees. However, our study also shows that only the best kernel methods can compete with a simple rule-based approach when the evaluation prevents information leakage between training and test corpora. Our results further reveal that the F-score of many approaches drops significantly if no corpus-specific parameter optimization is applied and that methods reaching a good AUC score often perform much worse in terms of F-score. We conclude that for most kernels no sensible estimation of PPI extraction performance on new text is possible, given the current heterogeneity in evaluation data. Nevertheless, our study shows that three

  17. A comprehensive benchmark of kernel methods to extract protein-protein interactions from literature.

    Directory of Open Access Journals (Sweden)

    Domonkos Tikk

    Full Text Available The most important way of conveying new findings in biomedical research is scientific publication. Extraction of protein-protein interactions (PPIs reported in scientific publications is one of the core topics of text mining in the life sciences. Recently, a new class of such methods has been proposed - convolution kernels that identify PPIs using deep parses of sentences. However, comparing published results of different PPI extraction methods is impossible due to the use of different evaluation corpora, different evaluation metrics, different tuning procedures, etc. In this paper, we study whether the reported performance metrics are robust across different corpora and learning settings and whether the use of deep parsing actually leads to an increase in extraction quality. Our ultimate goal is to identify the one method that performs best in real-life scenarios, where information extraction is performed on unseen text and not on specifically prepared evaluation data. We performed a comprehensive benchmarking of nine different methods for PPI extraction that use convolution kernels on rich linguistic information. Methods were evaluated on five different public corpora using cross-validation, cross-learning, and cross-corpus evaluation. Our study confirms that kernels using dependency trees generally outperform kernels based on syntax trees. However, our study also shows that only the best kernel methods can compete with a simple rule-based approach when the evaluation prevents information leakage between training and test corpora. Our results further reveal that the F-score of many approaches drops significantly if no corpus-specific parameter optimization is applied and that methods reaching a good AUC score often perform much worse in terms of F-score. We conclude that for most kernels no sensible estimation of PPI extraction performance on new text is possible, given the current heterogeneity in evaluation data. Nevertheless, our study

  18. Evaluation of protein extraction protocols for 2DE in marine ecotoxicoproteomics.

    Science.gov (United States)

    Wu, Huifeng; Ji, Chenglong; Wei, Lei; Zhao, Jianmin

    2013-11-01

    In ecotoxicoproteomics, an accurate and reproducible extraction of proteins is a critical step for 2DE analysis and further protein identification using MS. The criteria for the assessment of protein extraction quality include protein yield, protein spots resolved in a 2DE gel, matched protein spots in replicate gels, reproducibility, and compatibility with MS. In this work, we evaluated three protein extraction systems, straightforward lysis buffer, trichloroacetic acid-acetone, and TRIzol reagent with some modifications, for the protein extraction from three animal species including mussel Mytilus galloprovincialis, flounder Paralichthys olivaceus, and polychaete Nereis diversicolor used in marine ecotoxicology. Our results indicated that these methods could extract significantly different protein profiles. The method using TRIzol reagent resulted in the most matched protein spots resolved in four replicate 2DE gels and highest reproducibilities for the gill of M. galloprovincialis and liver of P. olivaceus. However, a modified trichloroacetic acid-acetone solvent system was best for the whole soft tissue of N. diversicolor. This work provides the fundamental information of the extraction quality of protein extraction protocols from different marine animals, which may facilitate the selection of a suitable protein extraction protocol for ecotoxicoproteomics.

  19. Extraction, composition and functional properties of pennycress (Thlaspi arvense L.) press cake protein

    Science.gov (United States)

    This study compared two methods for extracting the protein in pennycress (Thlaspi arvense L.) press cake and determined the composition and functional properties of the protein products. Proteins in pennycress press cake were extracted by using the conventional alkali solubilization-acid precipitati...

  20. Challenges of protein extraction from recalcitrant plant tissues for proteomics

    Science.gov (United States)

    Proteins play an important role in several biological processes. Proteomics encompasses basically four principal applications, namely protein mining, protein expression profiling, protein-network mapping and mapping of protein modifications. The results in these applications depend mostly on the c...

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

    Science.gov (United States)

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

    2012-01-12

    Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

  2. Improving Jatropha curcas seed protein recovery by using counter current multistage extraction

    NARCIS (Netherlands)

    Lestari, D.; Mulder, W.J.; Sanders, J.P.M.

    2010-01-01

    Jatropha curcas seed press cake contains 23 wt% proteins (dry basis). Due to the toxic compounds in Jatropha, we will use the proteins for non-food applications. Related to non-food applications, an efficient protein extraction to obtain a high protein recovery and high protein concentration with go

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

    NARCIS (Netherlands)

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

    2017-01-01

    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.

  4. Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls.

    Science.gov (United States)

    Yadav, Sunita; David, Anisha; Baluška, František; Bhatla, Satish C

    2013-03-01

    Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25-80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.

  5. Assessing the impact of protein extraction methods for human gut metaproteomics.

    Science.gov (United States)

    Zhang, Xu; Li, Leyuan; Mayne, Janice; Ning, Zhibin; Stintzi, Alain; Figeys, Daniel

    2017-07-10

    Metaproteomics is a promising methodology for the functional characterizations of the gut microbiome. However, the performance of metaproteomic analysis is affected by protein extraction protocols in terms of the amount of protein recovered and the relative abundance of different bacteria observed in microbiome. Currently, there is a lack of consistency on protein extraction methods in published metaproteomics studies. Here we evaluated the effects of different protein extraction methods on human fecal metaproteome characterizations. We found that sodium dodecyl sulfate (SDS)-based lysis buffer obtained higher protein yields and peptide/protein group identifications compared to urea and the non-ionic detergent-based B-Per buffer. The addition of bead beating to any of the extraction buffers increased both protein yields and protein identifications. As well, bead beating led to a significant increase of the relative abundances of Firmicutes and Actinobacteria. We also demonstrated that ultrasonication, another commonly used mechanical disruption approach, performed even better than bead beating for gut microbial protein extractions. Importantly, proteins of the basic metabolic pathways showed significantly higher relative abundances when using ultrasonication. Overall, these results demonstrate that protein extraction protocols markedly impact the metaproteomic results and recommend a protein extraction protocol with both SDS and ultrasonication for metaproteomic studies. The gut microbiome is emerging as an important factor influencing human health. Metaproteomics is promising for advancing the understanding of the functional roles of the microbiome in disease. However, metaproteomics suffers from a lack of consistent sample preparation procedures. In the present study, protein extraction protocols for fecal microbiome samples were evaluated for their effects on protein yields, peptide identifications, protein group identifications, taxonomic compositions and

  6. Osteocalcin and matrix GLA protein in developing teleost teeth: identification of sites of mRNA and protein accumulation at single cell resolution.

    Science.gov (United States)

    Ortiz-Delgado, J B; Simes, D C; Gavaia, P; Sarasquete, C; Cancela, M L

    2005-08-01

    In this study, the tissue distribution and accumulation of osteocalcin or bone Gla protein (BGP) and matrix Gla protein (MGP) were determined during tooth development in a teleost fish, Argyrosomus regius. In this species, the presence of BGP and MGP mRNA in teeth was revealed by in situ hybridization. mRNA for BGP was detected in the odontoblasts as well as in its cytoplasmic processes emerging through dentinal tubules, while mRNA for MGP was expressed in the enamel portion within the apical portion of the elongated cell bodies of enameloblasts, adjacent to the root of the teeth as well as in cells within the pulpal space. Immunolocalization of BGP and MGP demonstrated that these proteins accumulate mainly in the mineralized dentin or in enameloblastic processes, confirming in situ hybridization results. In this study, we examined for the first time the localization of both BGP and MGP gene expression and protein accumulation within the different regions of the vertebrate tooth. We clearly demonstrated that although the overall pattern of BGP and MGP gene expression and protein accumulation in A. regius teeth was in general agreement to what is known for other vertebrates such as rats or rodents, our study provided novel information and highlighted some species-differences between fish and higher vertebrates.

  7. Niemann-Pick disease type C2 protein induces triglyceride accumulation in silkworm and mammalian cell lines.

    Science.gov (United States)

    Adachi, Tatsuo; Ishii, Kenichi; Matsumoto, Yasuhiko; Hayashi, Yohei; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-04-01

    Silkworm haemolymph induced both the cessation of growth and an increase in triglyceride (triacylglycerol) storage in BmN4 cells. We purified the growth inhibitory factor from the silkworm haemolymph and identified this protein as the Bombyx mori PP (promoting protein), an orthologue of NPC2 (Niemann-Pick disease type C2) protein. Recombinant silkworm NPC2 inhibited cellular proliferation and increased triglyceride accumulation in BmN4 cells. Injection of either the recombinant protein or antiserum of NPC2 into living silkworms increased or decreased respectively triglyceride levels in the fat body. A mutation that depletes the cholesterol-binding capacity did not abolish the activity of NPC2. We further revealed that NPC2 induced the phosphorylation of AMPK (AMP-activated protein kinase) and that an AMPK inhibitor suppressed NPC2-dependent triglyceride accumulation. These findings suggest that NPC2 induces triglyceride accumulation via the activation of AMPK independently of its cholesterol-binding capacity in the silkworm.

  8. How Integrated Management Strategies Promote Protein Quality of Cotton Embryos: High Levels of Soil Available N, N Assimilation and Protein Accumulation Rate.

    Science.gov (United States)

    Yang, HongKun; Meng, YaLi; Chen, BingLin; Zhang, XingYue; Wang, YouHua; Zhao, WenQing; Zhou, ZhiGuo

    2016-01-01

    Cottonseed is widely used as a source of ruminant feed and for industrial purposes. Therefore, there is a tremendous need to improve the nutritional value of cotton embryos. In this study, a conventional management (CM) and two integrated cotton management strategies (IMS1, IMS2) were performed at two soil fertility levels to study the relationships among soil N, N assimilation, embryonic protein accumulation and protein quality. The levels of proteins, essential amino acids, and semi-essential amino acids, especially those of glutamate, lysine, and methionine, were higher in IMS1 and IMS2 embryos than in CM embryos. These changes were significantly positively correlated with the soil-available N content, glutamine synthetase activity and peak value of protein accumulation rate and were negatively correlated with the free amino acid level. These results illustrated that integrated management strategies, especially the rates and timing of N application, raise the level of soil available N, which is beneficial for N assimilation in developing cotton embryos. The protein content was limited by the rate of protein accumulation rather than by the free amino acid content. The combination of target yield fertilization, a growth-driven N application schedule, a high plant density and the seedling raising with bio-organic fertilizer can substantially improve protein quality in cotton embryos, especially at a soil with low soil organic matter and total nitrogen.

  9. Metformin reduces lipid accumulation in macrophages by inhibiting FOXO1-mediated transcription of fatty acid-binding protein 4

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jun [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Ren, Pingping; Zhang, Lin [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Wang, Xing Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States); Chen, Li [Qilu Hospital, Shandong University, Jinan, Shandong (China); Shen, Ying H., E-mail: hyshen@bcm.edu [Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX (United States); Texas Heart Institute at St. Luke' s Episcopal Hospital, Houston, TX (United States)

    2010-02-26

    Objective: The accumulation of lipids in macrophages contributes to the development of atherosclerosis. Strategies to reduce lipid accumulation in macrophages may have therapeutic potential for preventing and treating atherosclerosis and cardiovascular complications. The antidiabetic drug metformin has been reported to reduce lipid accumulation in adipocytes. In this study, we examined the effects of metformin on lipid accumulation in macrophages and investigated the mechanisms involved. Methods and results: We observed that metformin significantly reduced palmitic acid (PA)-induced intracellular lipid accumulation in macrophages. Metformin promoted the expression of carnitine palmitoyltransferase I (CPT-1), while reduced the expression of fatty acid-binding protein 4 (FABP4) which was involved in PA-induced lipid accumulation. Quantitative real-time PCR showed that metformin regulates FABP4 expression at the transcriptional level. We identified forkhead transcription factor FOXO1 as a positive regulator of FABP4 expression. Inhibiting FOXO1 expression with FOXO1 siRNA significantly reduced basal and PA-induced FABP4 expression. Overexpression of wild-type FOXO1 and constitutively active FOXO1 significantly increased FABP4 expression, whereas dominant negative FOXO1 dramatically decreased FABP4 expression. Metformin reduced FABP4 expression by promoting FOXO1 nuclear exclusion and subsequently inhibiting its activity. Conclusions: Taken together, these results suggest that metformin reduces lipid accumulation in macrophages by repressing FOXO1-mediated FABP4 transcription. Thus, metformin may have a protective effect against lipid accumulation in macrophages and may serve as a therapeutic agent for preventing and treating atherosclerosis in metabolic syndrome.

  10. Differential protein accumulations in isolates of the strawberry wilt pathogen Fusarium oxysporum f. sp. fragariae differing in virulence.

    Science.gov (United States)

    Fang, Xiangling; Barbetti, Martin J

    2014-08-28

    This study was conducted to define differences in Fusarium oxysporum f. sp. fragariae (Fof) isolates with different virulence efficiency to strawberry at the proteome level, in combination with their differences in mycelial growth, conidial production and germination. Comparative proteome analyses revealed substantial differences in mycelial proteomes between Fof isolates, where the 54 differentially accumulated protein spots were consistently over-accumulated or exclusively in the highly virulent isolate. These protein spots were identified through MALDI-TOF/TOF mass spectrometry analyses, and the identified proteins were mainly related to primary and protein metabolism, antioxidation, electron transport, cell cycle and transcription based on their putative functions. Proteins of great potential as Fof virulence factors were those involved in ubiquitin/proteasome-mediated protein degradation and reactive oxygen species detoxification; the hydrolysis-related protein haloacid dehalogenase superfamily hydrolase; 3,4-dihydroxy-2-butanone 4-phosphate synthase associated with riboflavin biosynthesis; and those exclusive to the highly virulent isolate. In addition, post-translational modifications may also make an important contribution to Fof virulence. F. oxysporum f. sp. fragariae (Fof), the causal agent of Fusarium wilt in strawberry, is a serious threat to commercial strawberry production worldwide. However, factors and mechanisms contributing to Fof virulence remained unknown. This study provides knowledge of the molecular basis for the differential expression of virulence in Fof, allowing new possibilities towards developing alternative and more effective strategies to manage Fusarium wilt. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Effect of chickpea aqueous extracts, organic extracts, and protein concentrates on cell proliferation.

    Science.gov (United States)

    Girón-Calle, Julio; Vioque, Javier; del Mar Yust, María; Pedroche, Justo; Alaiz, Manuel; Millán, Francisco

    2004-01-01

    Pulses should be part of a healthy diet, and it is also becoming clear that they have health-promoting effects. Nevertheless, most studies on the bioactive or health-promoting properties of pulses have been carried out using soybeans. We have studied cell growth-regulating properties, which may be responsible for anti-cancer properties, in chickpea seeds. Chickpea seeds are a staple in the traditional diet of many Mediterranean, Asian, and South and Central American countries. In addition, chickpea seeds have industrial applications since they can be used for the preparation of protein concentrates and isolates. The cell lines Caco-2 (epithelial intestinal) and J774 (macrophages) have been exposed to chickpea seed extracts and protein preparations in order to screen the different chickpea fractions for effects on cell growth. Both cell growth-promoting and cell growth-inhibiting effects were found. Most interestingly, a fraction soluble in ethanol and acetone specifically and almost completely inhibited the growth of Caco-2 cells exhibiting a cancerous phenotype. It is concluded that chickpea seeds are a source of bioactive components and deserve further study for their possible anti-cancer effect.

  12. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  13. Protein biomarkers of external root resorption: A new protein extraction protocol. Are we going in the right direction?

    OpenAIRE

    2014-01-01

    OBJECTIVE: The aim of this study is to determine a protocol of gingival crevicular fluid protein extraction used for the first dimension of 2-DE gels. It also aims at conducting a review on the current candidates for protein markers of this pathology, all of which may be used to prevent the disease. METHODS: Gingival crevicular fluid was collected from two groups of 60 patients each, with and without external root resorption. Samples were extracted by means of various methods of protein extra...

  14. Labile proteins accumulated in damaged hair upon permanent waving and bleaching treatments

    National Research Council Canada - National Science Library

    Inoue, Takafumi; Ito, Mayumi; Kizawa, Kenji

    2002-01-01

    .... These results suggest that a portion of the stable proteins in normal hair was transformed into labile proteins upon permanent waving and bleaching treatments. Consequently, permed and bleached hair tends to release the resultant labile proteins.

  15. Extraction and characterization of protein from Irish brown seaweed Ascophyllum nodosum.

    Science.gov (United States)

    Kadam, Shekhar U; Álvarez, Carlos; Tiwari, Brijesh K; O'Donnell, Colm P

    2017-09-01

    This study investigates traditional and non-conventional methods of extraction of protein from Irish brown seaweed A. nodosum. Acid, alkali, combined acid-alkali with and without ultrasound pretreatment were investigated for extraction of protein from A. nodosum. Molecular weight of protein was determined using high performance size exclusion chromatography and amino acid profiling was carried out using an amino acid analyzer. Combination of first acid and then alkali extraction was found to be the most efficient method of extraction among all methods investigated (59% of recovery); followed by single step of alkali extraction assisted with ultrasound (68.4μm) which was able to extract 57% of total protein. Alkaline extraction was shown to yield the best protein/algae liquefaction ratio (1.28). This can be attributed to the release of polysaccharide complexes first by acid and then solubilization of proteins by alkali solvent. The molecular weight of extracted protein was found to be relatively low, in the range of 2-4kDa average MW. The alkali method of extraction was found to be optimum for extraction of amino acids from A. nodosum. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Optimising the Use of TRIzol-extracted Proteins in Surface Enhanced Laser Desorption/ Ionization (SELDI Analysis

    Directory of Open Access Journals (Sweden)

    Perlaky Laszlo

    2006-03-01

    Full Text Available Abstract Background Research with clinical specimens is always hampered by the limited availability of relevant samples, necessitating the use of a single sample for multiple assays. TRIzol is a common reagent for RNA extraction, but DNA and protein fractions can also be used for other studies. However, little is known about using TRIzol-extracted proteins in proteomic research, partly because proteins extracted from TRIzol are very resistant to solubilization. Results To facilitate the use of TRIzol-extracted proteins, we first compared the ability of four different common solubilizing reagents to solubilize the TRIzol-extracted proteins from an osteosarcoma cell line, U2-OS. Then we analyzed the solubilized proteins by Surface Enhanced Laser Desorption/ Ionization technique (SELDI. The results showed that solubilization of TRIzol-extracted proteins with 9.5 M Urea and 2% CHAPS ([3-[(3-cholamidopropyl-dimethylammonio]propanesulfonate] (UREA-CHAPS was significantly better than the standard 1% SDS in terms of solubilization efficiency and the number of detectable ion peaks. Using three different types of SELDI arrays (CM10, H50, and IMAC-Cu, we demonstrated that peak detection with proteins solubilized by UREA-CHAPS was reproducible (r > 0.9. Further SELDI analysis indicated that the number of ion peaks detected in TRIzol-extracted proteins was comparable to a direct extraction method, suggesting many proteins still remain in the TRIzol protein fraction. Conclusion Our results suggest that UREA-CHAPS performed very well in solubilizing TRIzol-extracted proteins for SELDI applications. Protein fractions left over after TRIzol RNA extraction could be a valuable but neglected source for proteomic or biochemical analysis when additional samples are not available.

  17. Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

    Science.gov (United States)

    El Asmar, Zeina; Terrand, Jérome; Jenty, Marion; Host, Lionel; Mlih, Mohamed; Zerr, Aurélie; Justiniano, Hélène; Matz, Rachel L; Boudier, Christian; Scholler, Estelle; Garnier, Jean-Marie; Bertaccini, Diego; Thiersé, Danièle; Schaeffer, Christine; Van Dorsselaer, Alain; Herz, Joachim; Bruban, Véronique; Boucher, Philippe

    2016-03-04

    The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

  18. Anti-Cancer Effects of Protein Extracts from Calvatia lilacina, Pleurotus ostreatus and Volvariella volvacea

    Directory of Open Access Journals (Sweden)

    Jin-Yi Wu

    2011-01-01

    Full Text Available Calvatia lilacina (CL, Pleurotus ostreatus (PO and Volvariella volvacea (VV are widely distributed worldwide and commonly eaten as mushrooms. In this study, cell viabilities were evaluated for a human colorectal adenocarcinoma cell line (SW480 cells and a human monocytic leukemia cell line (THP-1 cells. Apoptotic mechanisms induced by the protein extracts of PO and VV were evaluated for SW480 cells. The viabilities of THP-1 and SW480 cells decreased in a concentration-dependent manner after 24 h of treatment with the protein extracts of CL, PO or VV. Apoptosis analysis revealed that the percentage of SW480 cells in the SubG1 phase (a marker of apoptosis was increased upon PO and VV protein-extract treatments, indicating that oligonucleosomal DNA fragmentation existed concomitantly with cellular death. The PO and VV protein extracts induced reactive oxygen species (ROS production, glutathione (GSH depletion and mitochondrial transmembrane potential (ΔΨm loss in SW480 cells. Pretreatment with N-acetylcysteine, GSH or cyclosporine A partially prevented the apoptosis induced by PO protein extracts, but not that induced by VV extracts, in SW480 cells. The protein extracts of CL, PO and VV exhibited therapeutic efficacy against human colorectal adenocarcinoma cells and human monocytic leukemia cells. The PO protein extracts induced apoptosis in SW480 cells partially through ROS production, GSH depletion and mitochondrial dysfunction. Therefore, the protein extracts of these mushrooms could be considered an important source of new anti-cancer drugs.

  19. Purple Tea and Its Extract Suppress Diet-induced Fat Accumulation in Mice and Human Subjects by Inhibiting Fat Absorption and Enhancing Hepatic Carnitine Palmitoyltransferase Expression.

    Science.gov (United States)

    Shimoda, Hiroshi; Hitoe, Shoketsu; Nakamura, Seikou; Matsuda, Hisashi

    2015-06-01

    A number of clinical trials have been completed using green tea and black tea to investigate their effect in controlling weight in overweight adults. The results of these investigations, however, have often been contradictory, with some trials reporting positive effects of tea supplementation and some trials reporting no effect. As a result, the use of these teas for weight loss is controversial. Purple tea is a variety of green tea developed in Kenya (called TRFK306), which in addition to certain tea constituents found in green tea, also contains anthocyanins. The major constituents in the leaves of purple tea are caffeine, theobromine, epigallocatechin (ECG), epigallocatechin gallate (EGCG) and 1,2-di-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-β-D-glucose (GHG). We investigated the efficacy of purple tea extract (PTE) on diet-induced fat accumulation in mice. PTE administration (200 mg/kg) significantly suppressed body weight gain, liver weight, abdominal fat and triglycerides in serum and liver. Protein expression of carnitine palmitoyltransferase (CPT) 1A was also enhanced. In olive oil loaded mice, PTE (100 mg/kg) and caffeine (25 mg/kg) suppressed fat absorption. PTE (10 μg/mL) and GHG (10 μg/mL) also enhanced protein expression of CPT1A in HepG2 hepatoma. Moreover, 4-week daily consumption of purple tea drink in humans improved obesity parameters compared to baseline, including body weight (79.9 ± 3.1 kg vs 80.8 ± 3.2, pweight gain by suppression of fat absorption and enhancement of hepatic fat metabolism.

  20. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans.

    Science.gov (United States)

    Andralojc, Karolina M; Campbell, Anne C; Kelly, Ashley L; Terrey, Markus; Tanner, Paige C; Gans, Ian M; Senter-Zapata, Michael J; Khokhar, Eraj S; Updike, Dustin L

    2017-02-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.

  1. Extracting Tenebrio molitor protein while preventing browning: effect of pH and NaCl on protein yield

    NARCIS (Netherlands)

    Yi, L.; Boekel, van T.; Lakemond, C.M.M.

    2017-01-01

    The potential of insects as an alternative protein source for food applications was investigated by studying the effect of pH and NaCl on extraction yield of water-soluble proteins from Tenebrio molitor, while preventing browning due to polyphenol oxidation. Minimum protein solubility (29.6%) was at

  2. Extracting Tenebrio molitor protein while preventing browning: effect of pH and NaCl on protein yield

    NARCIS (Netherlands)

    Yi, L.; Boekel, van T.; Lakemond, C.M.M.

    2016-01-01

    The potential of insects as an alternative protein source for food applications was investigated by studying the effect of pH and NaCl on extraction yield of water-soluble proteins from Tenebrio molitor, while preventing browning due to polyphenol oxidation. Minimum protein solubility (29.6%) was at

  3. An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays

    Directory of Open Access Journals (Sweden)

    Guo Huifang

    2012-09-01

    Full Text Available Abstract Introduction Protein extraction from formalin-fixed paraffin-embedded (FFPE tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. Results Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples. The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p Conclusions With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.

  4. Efficient extraction of olive pulp and stone proteins by using an enzyme-assisted method.

    Science.gov (United States)

    Vergara-Barberán, María; Lerma-García, María Jesús; Herrero-Martínez, José Manuel; Simó-Alfonso, Ernesto Francisco

    2014-07-01

    An efficient protein extraction protocol for proteins from olive pulp and stone by using enzymes was developed. For this purpose, different parameters that affect the extraction process, such as enzyme type and content, pH, and extraction temperature and time, were tested. The influence of these factors on protein recovery was examined using the standard Bradford assay, while the extracted proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The best extraction conditions were achieved at pH 7.0 and 5% (v/v) Palatase® 20000 L (lipase) for pulp and Lecitase® Ultra (phospholipase) for stone proteins. The optimal extraction temperature and time were 30 and 40 °C for 15 min for pulp and stone tissues, respectively. Under these conditions, several protein extracts coming from olive fruits of different genetic variety were analyzed, their profiles being compared by SDS-PAGE. The developed enzyme-assisted extraction method showed faster extraction, higher recovery, and reduced solvent usage than the nonenzymatic methods previously described in the literature. In the case of stone proteins, different electrophoretic profiles and band intensities were obtained that could be helpful to distinguish samples according to their genetic variety.

  5. Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

    Science.gov (United States)

    Shi, Shan-Rong; Liu, Cheng; Balgley, Brian M; Lee, Cheng; Taylor, Clive R

    2006-06-01

    A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    at position 124 in mature TGFBIp leads to granular corneal dystrophy type 2 (GCD2). Homozygous GCD2 cases develop massive protein accumulation early in life whereas heterozygous GCD2 cases become affected much later and generally with a much less severe outcome. However, if heterozygous GCD2 patients undergo...... laser-assisted in situ keratomileusis (LASIK) surgery protein accumulation is accelerated and they develop massive protein accumulations a few years after surgery. Here, we present the protein profile of aggregate-containing corneal tissue from GCD2 patients with a history of LASIK surgery using LC......-MS/MS. Label-free quantification of corneal extracellular matrix proteins showed accumulation of TGFBIp. This was supported by 2DE and immunoblotting against TGFBIp that revealed the accumulation of full-length TGFBIp. In addition, a high molecular weight TGFBIp complex was more apparent in GCD2 patients after...

  7. Characterization of rice starch and protein obtained by a fast alkaline extraction method.

    Science.gov (United States)

    Souza, Daiana de; Sbardelotto, Arthur Francisco; Ziegler, Denize Righetto; Marczak, Ligia Damasceno Ferreira; Tessaro, Isabel Cristina

    2016-01-15

    This study evaluated the characteristics of rice starch and protein obtained by a fast alkaline extraction method on rice flour (RF) derived from broken rice. The extraction was conducted using 0.18% NaOH at 30°C for 30min followed by centrifugation to separate the starch rich and the protein rich fractions. This fast extraction method allowed to obtain an isoelectric precipitation protein concentrate (IPPC) with 79% protein and a starchy product with low protein content. The amino acid content of IPPC was practically unchanged compared to the protein in RF. The proteins of the IPPC underwent denaturation during extraction and some of the starch suffered the cold gelatinization phenomenon, due to the alkaline treatment. With some modifications, the fast method can be interesting in a technological point of view as it enables process cost reduction and useful ingredients obtention to the food and chemical industries.

  8. Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

    Directory of Open Access Journals (Sweden)

    Witzel Katja

    2011-12-01

    Full Text Available Abstract Proteins in the plant apoplast are essential for many physiological processes. We have analysed and compared six different infiltration solutions for proteins contained in the apoplast to recognize the most suitable method for leaves and to establish proteome maps for each extraction. The efficiency of protocols was evaluated by comparing the protein patterns resolved by 1-DE and 2-DE, and revealed distinct characteristics for each infiltration solution. Nano-LC-ESI-Q-TOF MS analysis of all fractions was applied to cover all proteins differentially extracted by infiltration solutions and led to the identification of 328 proteins in total in apoplast preparations. The predicted subcellular protein localisation distinguished the examined infiltration solutions in those with high or low amounts of intracellular protein contaminations, and with high or low quantities of secreted proteins. All tested infiltration solution extracted different subsets of proteins, and those implications on apoplast-specific studies are discussed.

  9. Potato tuber proteomics: Comparison of two complementary extraction methods designed for 2-DE of acidic proteins

    NARCIS (Netherlands)

    Delaplace, P.; Wal, van der F.; Dierick, J.F.; Cordewener, J.H.G.; Fauconnier, M.L.; Jardin, du P.; America, A.H.P.

    2006-01-01

    Two protein extraction procedures were tested in order to remove interfering compounds prior to 2-DE of potato tubers. These methods using SDS lysis buffer and phenol-phase extraction were compared regarding the quality of the resulting 2-D gel. While the resolution of SDS extracts on semipreparativ

  10. Subcellular clearance and accumulation of Huntington disease protein: A mini-review

    Directory of Open Access Journals (Sweden)

    Ting eZhao

    2016-04-01

    Full Text Available Huntington’s disease (HD is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ tract in the N-terminal region of mutant huntingtin (mHtt. As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies of examining mHtt clearance and accumulation in different subcellular regions

  11. Sorghum Protein Extraction by Sonication and Its Relationship to Ethanol Fermentation

    Science.gov (United States)

    The objectives of this research were to develop a rapid method for extracting proteins from mashed and non-mashed sorghum meal using sonication (ultrasound), and to determine the relationships between the levels of extractable proteins and ethanol fermentation. Nine grain sorghum samples with a bro...

  12. Chaperone potential of Pulicaria undulata extract in preventing aggregation of stressed proteins.

    Science.gov (United States)

    Ghahghaei, Arezou; Valizadeh, Jafar; Nazari, Shahrzad; Ravandeh, Mehdi

    2014-06-01

    This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.

  13. Critical parameters in cost-effective alkaline extraction for high protein yield from leaves

    NARCIS (Netherlands)

    Zhang, C.; Sanders, J.P.M.; Bruins, M.E.

    2014-01-01

    Leaves are potential resources for feed or food, but their applications are limited due to a high proportion of insoluble protein and inefficient processing. To overcome these problems, parameters of alkaline extraction were evaluated using green tea residue (GTR). Protein extraction could be maximi

  14. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    Science.gov (United States)

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

  15. Chemical optimization of protein extraction from sweet potato (Ipomoea batatas) peel

    Science.gov (United States)

    Proteins isolated from sweet potatoes (Ipomoea batatas) have been shown to possess antidiabetic, antioxidant, and antiproliferative properties. The objective of this study was to chemically optimize a process for extracting proteins from sweet potato peel. The extraction procedure involved mixing pe...

  16. Adhesive performance of sorghum protein extracted from sorghum DDGS and flour

    Science.gov (United States)

    Distillers dried grains with solubles (DDGS) is the main co-product from grain-based ethanol production. The objective of this research was to compare the adhesive performance of three types of sorghum proteins: acetic acid-extracted sorghum protein from DDGS (PI), aqueous ethanol-extracted sorghum ...

  17. Characterisation of kiwifruit and asparagus enzyme extracts, and their activities toward meat proteins.

    Science.gov (United States)

    Ha, Minh; Bekhit, Alaa El-Din; Carne, Alan; Hopkins, David L

    2013-01-15

    Two plant enzyme extracts from kiwifruit and asparagus were evaluated for their ability to hydrolyse commercially available substrates and proteins present in both beef connective tissue and topside myofibrillar extracts. The results show significant differences in protease activity depending on the assay used. Protease assays with connective tissue and meat myofibrillar extracts provide a more realistic evaluation of the potential of the enzymes for application in meat tenderization. Overall, the kiwifruit protease extract was found to be more effective at hydrolysing myofibrillar and collagen proteins than the asparagus protease extract. The two protease extracts appeared to target meat myofibrillar and collagen proteins differently, suggesting the potential of a synergistic effect of these proteases in improving the tenderness of specific cuts of meat, based on their intrinsic protein composition.

  18. Effects of Sallropus androgynus (katuk leaf extract on growth, fat accumulation and fecal microorganisms in broiler chickens

    Directory of Open Access Journals (Sweden)

    U Santoso

    2001-12-01

    Full Text Available A study was conducted to determine the effects of Sauropus androgynus leaf extract on growth, carcass quality and the number of fecal microorganisms in broiler chickens. Forty-eight male Arbor Acres broiler chickens (21-d-old obtained from a commercial hatchery were used in the present study. Experiment consisted of four treatment groups with four pen replicates of three broilers allotted randomly to each dietary treatment from day 21-42 old. One group was the control with no additional Sauropus androgynus leaf extract (SAE (P0, and other three groups were given drinking water supplemented with 1.5 g (PI, 3.0 g (P2 or 4.5 g SAE/l water (P3. The diet used was a commercial mix (Crude protein: 19% and Metabolizable Energy 3200 kcal/kg. Feed intake significantly reduced in P2 or P3 as compared with the control (P<0.05. A decrease in feed conversion ratio was observed in treatment groups as compared with Po (P<0.05. Abdominal fat, neck fat and liver fat content were significantly reduced by SAE (P<0.05, while carcass fat content was not significantly different. Number of fecal Escherichia coli in P1 or P3 (P<0,01 and fecal Streptococcus sp and Salmonella sp. were significantly (P<0,01 reduced by SAE supplementation as compared with the control, while fecal Bacillus subtilis in P2 and Lactobacillus sp. in P1 (P<0,01 were significantly higher as compared with other groups. SAE (P2 or P3 also significantly improved meat taste, shank color but lowered meat color (P<0.05 as compared with the control group. It was found that the number of fecal Streptococcus sp was suitable to predict abdominal fat. In conclusion, the inclusion of SAE at 4.5 g/l drinking water resulted in the best performance and carcass quality.

  19. A protein related to prokaryotic UMP kinases is involved in psaA/B transcript accumulation in Arabidopsis.

    Science.gov (United States)

    Hein, Paul; Stöckel, Jana; Bennewitz, Stefan; Oelmüller, Ralf

    2009-03-01

    Dpt1 (defect in p saA/B transcript accumulation 1) is a novel photosystem (PS) I mutant in Arabidopsis. dpt1 mutants fail to grow photoautotrophically, and are impaired in the accumulation of psaA/B transcripts while the transcript levels for the remaining PSI subunits, for subunits of the PSII, the cyt-b ( 6 )/f-complex, and the ribulose-1,5-bisphosphate carboxylase are comparable to the wild type. In-organello run-on transcription assays demonstrate that the lower psaA/B transcript abundance in dpt1-1 is not caused by the inability to transcribe the psaA/psaB/rps14 operon. psaA/B transcripts in the mutant are associated with polyribosomes and translated. Thus, the mutation affects post-transcriptional processes specific for psaA/B. The dpt1 gene was isolated by map-based cloning. The protein is localized in the stroma of the chloroplast and exhibits striking similarities to UMP kinases of prokaryotic origin. Our results show that the nuclear encoded protein Dpt1 is essential for retaining photosynthetic activity in higher plant chloroplasts and involved in post-transcriptional steps of psaA/B transcript accumulation. We discuss that Dpt1 may be a bifunctional protein that couples the pyrimidine metabolism to the photosynthetic electron transport.

  20. Transgenic soya bean seeds accumulating β-carotene exhibit the collateral enhancements of oleate and protein content traits.

    Science.gov (United States)

    Schmidt, Monica A; Parrott, Wayne A; Hildebrand, David F; Berg, R Howard; Cooksey, Amanda; Pendarvis, Ken; He, Yonghua; McCarthy, Fiona; Herman, Eliot M

    2015-05-01

    Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 μg β carotene g(-1) dry seed weight with a desirable 12:1 ratio of β to α. The β carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated β-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the mid-maturation β-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated β-carotene, oleic acid and protein traits in the β-carotene soya beans confer a substantial additive nutritional quality to soya beans. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Comparison of Three Methods of Protein Extraction from Dermatophagoides Pteronyssinus for Two-dimensional Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    Jin-lu Sun; Hong-yu Zhang; Zhi-yi Guo; Wan-tao Ying; Xiao-hong Qian; Jing-lan Wang

    2009-01-01

    Objective To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis (2-DE) analysis. Methods The extracts of Dermatophagoides pteronyssinus were prepared with Coca's solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid (BCA) assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis. Results The concentrations of extracted protein by methods of Coca's solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight (LMW) range could be detected with the Coca's solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight (MMW) protein spots were absent. Several MMW protein spots (174-178 kD and 133 kD) and more LMW protein spots were detected with Trizol reagent method. Conclusions Among Coca's solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.

  2. Evaluation of effect of alcoholic extract of heartwood of Pterocarpus marsupium on in vitro antioxidant, anti-glycation, sorbitol accumulation and inhibition of aldose reductase activity

    Directory of Open Access Journals (Sweden)

    Pankaj Gupta

    2017-07-01

    The alcoholic extract of P. marsupium across varying concentrations showed inhibitory effect as evident by IC50 on advanced glycation-end-products formation (55.39 μg/ml, sorbitol accumulation (151.00 μg/ml and rat kidney aldose reductase (195.88 μg/ml. The phytoextract also exhibited high phenolic and flavonoid contents with promising antioxidant potential against the antioxidant assays evaluated. The present investigation suggests that the phytoextract showed prominent antioxidant, antiglycation property and, inhibited accumulation of sorbitol and ALR enzyme, thus promising a beneficial role in reducing/delaying diabetic complications.

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

    Science.gov (United States)

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

    2016-11-05

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

  4. Overexpression of a stress-responsive U-box protein gene VaPUB affects the accumulation of resistance related proteins in Vitis vinifera 'Thompson Seedless'.

    Science.gov (United States)

    Jiao, Li; Zhang, Yali; Lu, Jiang

    2017-03-01

    Many U-box proteins have been identified and characterized as important factors against environmental stresses such as chilling, heat, salinity and pathogen attack in plant. Our previous research reported the cloning of a novel U-box protein gene VaPUB from Vitis amurensis 'Zuoshanyi' grape and suggested a function of it in related to cold stress in the model plant Arabidopsis system. In this study, the role of VaPUB in response to biotic and abiotic stress was further analyzed in the homologous grapevine system by studying the transcript regulation and the protein accumulation in VaPUB transgenic vines. The expression analysis assay shown that VaPUB was significantly up-regulated 6 h after cold treatment and as early as 2 h post inoculation with Plasmopara viticola, a pathogen causing downy mildew disease in grapevine. Over-expressing VaPUB in V. Vinifera 'Thompson Seedless' affected the microstructure of leaves. The proteome assay shown that the accumulation of pathogenesis-related protein PR10 and many proteins involved in carbon and energy metabolism, oxidation reaction and protein metabolism were significantly altered in transgenic vines. In comparison with wild type plants, the expression level of PR10 family genes was significantly decreased in VaPUB transgenic vines under P. viticola treatment or cold stress. Results from this study showed that the U-box protein gene PUB quickly responded to both biotic stress and abiotic stress and significantly influenced the accumulation of resistance related proteins in grapevine. Copyright © 2016. Published by Elsevier Masson SAS.

  5. Current Physical and SDS Extraction Methods Do Not Efficiently Remove Exosporium Proteins from Bacillus anthracis spores

    OpenAIRE

    Thompson, Brian M.; Binkley, Jana M; Stewart, George C.

    2011-01-01

    Biochemical studies of the outermost spore layers of the Bacillus cereus family are hindered by difficulties in efficient dispersal of the external spore layers and difficulties in dissociating protein complexes that comprise the exosporium layer. Detergent and physical methods have been utilized to disrupt the exosporium layer. Herein we compare commonly used SDS extraction buffers used to extract spore proteins and demonstrate the incomplete extractability of the exosporium layer by these m...

  6. Evaluation of extraction solutions for biochemical analyses of the proteins in rice grains.

    Science.gov (United States)

    Lang, Gang-hua; Kagiya, Yukari; Ohnishi-Kameyama, Mayumi; Kitta, Kazumi

    2013-01-01

    The influence of different extraction solutions on the proteins extracted from rice grains was investigated. The largest amounts of salt-soluble proteins were extracted with solutions supplemented with Tris-HCl at pH 8.0. Rice allergens were analyzed by multiplex immunodetection. Except for α-globulin extracted with the solutions at pH 8.0, which showed a low-molecular-weight band besides the main band, no significant solution-dependent difference among the allergens was found. Total proteins were extracted with four kinds of solution. The extraction of the basic subunit of glutelin was found to be SDS-dependent, and more protein was obtained with extraction solutions supplemented with SDS. The contents of α-globulin and α-amylase/trypsin inhibitors were higher in the extracts without SDS than with SDS. We conclude from the present data that, in order to obtain comparable data from rice grain salt-soluble and total protein analyses, differences in the protein extraction efficiency of solutions used should be taken into consideration.

  7. Improved proteomic analysis following trichloroacetic acid extraction of Bacillus anthracis spore proteins.

    Science.gov (United States)

    Deatherage Kaiser, Brooke L; Wunschel, David S; Sydor, Michael A; Warner, Marvin G; Wahl, Karen L; Hutchison, Janine R

    2015-11-01

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.

  8. Reduction of adipogenesis and lipid accumulation by Taraxacum officinale (Dandelion) extracts in 3T3L1 adipocytes: an in vitro study.

    Science.gov (United States)

    González-Castejón, Marta; García-Carrasco, Belén; Fernández-Dacosta, Raquel; Dávalos, Alberto; Rodriguez-Casado, Arantxa

    2014-05-01

    In this in vitro study, we have investigated the ability of Taraxacum officinale (dandelion) to inhibit adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes. HPLC analysis of the three plant extracts used in this study-leaf and root extracts and a commercial root powder-identified caffeic and chlorogenic acids as the main phenolic constituents. Oil Red O staining and triglyceride levels analysis showed decreased lipid and triglyceride accumulation, respectively. Cytotoxicity was assessed with the MTT assay showing non-toxic effect among the concentrations tested. DNA microarray analysis showed that the extracts regulated the expression of a number of genes and long non-coding RNAs that play a major role in the control of adipogenesis. Taken together, our results indicate that the dandelion extracts used in this study may play a significant role during adipogenesis and lipid metabolism, and thus, supporting their therapeutic interest as potential candidates for the treatment of obesity.

  9. pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.

    Science.gov (United States)

    Gong, Xiaoyan; Hurtado, Oscar; Wang, Baohua; Wu, Congqing; Yi, Mihwa; Giraldo, Martha; Valent, Barbara; Goodin, Michael; Farman, Mark

    2015-02-01

    As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

  10. Integrating kinetics with thermodynamics to study the alkaline extraction of protein from Caragana korshinskii Kom.

    Science.gov (United States)

    Zhong, Cheng; Zhou, Zhao; Zhang, Yu-Ming; Jia, Shi-Ru; Sun, Zhuo; Dale, Bruce E

    2014-09-01

    Extraction and recovery of protein from abundant plant biomass is one potential way to improve the economic feasibility of biorefineries. However, valorization of the protein fraction is challenging due to its low yield (kg protein extraction/kg biomass). In order to reveal the limiting operation parameters, the alkaline extraction process of protein from Caragana korshinskii Kom. was investigated by an integrative analysis of kinetics and thermodynamics. Both a two-site kinetic extraction model and a second-order model indicated that particle size is the most pivotal factor affecting protein extraction yield. In a two-site model, most proteins are extracted quickly from broken cells, while protein removal from the intact cells takes much longer; these are the faster and slower processes, respectively. A decrease of particle size from 20-40 to 60-80 mesh resulted in a decrease of C2 (protein yield in the slower process) from 14.02 to 7.32 mg g(-1), but a great increase of C1 (protein yield in the faster process) from 20.61 to 59.07 mg g(-1) . However, the protein yield was dominated by the faster process when the average particle size is under 80 mesh. The maximum initial extraction rate was 72.20 mg g(-1) min(-1) with the particle size of 60-80 mesh, almost ninefold of that with 20-40 mesh. Thermodynamic analysis revealed that the enthalpy change (ΔH) and entropy change (ΔS) in the protein extraction process were calculated as 21.08 kJ mol(-1) and 84.76 J K(-1), respectively. The standard free energy (ΔG) had a magnitude from -3.77 to -5.46, suggesting that the extraction process was spontaneous and physically feasible.

  11. Evaluation of Protein Extraction Methods for Proteomic Analysis of Non-Model Recalcitrant Plant Tissues

    OpenAIRE

    2012-01-01

    Plant tissues contain relatively low amounts of proteins whose extraction is often difficult due to the presence of interfering compounds such as rigid cellulosic cell wall, storage polysaccharides, lipids and other contaminants that can cause protein degradation or modification. Therefore it is important to optimize protein extraction and to establish a robust protocol for two-dimensional gel electrophoresis (2-DE) and downstream processing. In this study, acetone, trichloroacetic acid/aceto...

  12. Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation

    Science.gov (United States)

    Matukumalli, Suvarsha Rao; Tangirala, Ramakrishna; Rao, C. M.

    2017-01-01

    Proteins, made up of either single or multiple chains, are designed to carry out specific biological functions. We found an interesting example of a two-chain protein where administration of one of its chains leads to a diametrically opposite outcome than that reported for the full-length protein. Clusterin is a highly glycosylated protein consisting of two chains, α- and β-clusterin. We have investigated the conformational features, cellular localization, lipid accumulation, in vivo effects and histological changes upon administration of recombinant individual chains of clusterin. We demonstrate that recombinant α- and β-chains exhibit structural and functional differences and differ in their sub-cellular localization. Full-length clusterin is known to lower lipid levels. In contrast, we find that β-chain-treated cells accumulate 2-fold more lipid than controls. Interestingly, α-chain-treated cells do not show such increase. Rabbits injected with β-chain, but not α-chain, show ~40% increase in weight, with adipocyte hypertrophy, liver and kidney steatosis. Many, sometimes contrasting, roles are ascribed to clusterin in obesity, metabolic syndrome and related conditions. Our findings of differential localization and activities of individual chains of clusterin should help in understanding better the roles of clusterin in metabolism. PMID:28120874

  13. The D1 and D2 proteins of dinoflagellates: unusually accumulated mutations which influence on PSII photoreaction.

    Science.gov (United States)

    Iida, Satoko; Kobiyama, Atsushi; Ogata, Takehiko; Murakami, Akio

    2008-01-01

    Plastid encoded genes of the dinoflagellates are rapidly evolving and most divergent. The importance of unusually accumulated mutations on structure of PSII core protein and photosynthetic function was examined in the dinoflagellates, Symbiodinium sp. and Alexandrium tamarense. Full-length cDNA sequences of psbA (D1 protein) and psbD (D2 protein) were obtained and compared with the other oxygen-evolving photoautotrophs. Twenty-three amino acid positions (7%) for the D1 protein and 34 positions (10%) for the D2 were mutated in the dinoflagellates, although amino acid residues at these positions were conserved in cyanobacteria, the other algae, and plant. Many mutations were likely to distribute in the N-terminus and the D-E interhelical loop of the D1 protein and helix B of D2 protein, while the remaining regions were well conserved. The different structural properties in these mutated regions were supported by hydropathy profiles. The chlorophyll fluorescence kinetics of the dinoflagellates was compared with Synechocystis sp. PCC6803 in relation to the altered protein structure.

  14. Enzymatic Pre-Treatment Increases the Protein Bioaccessibility and Extractability in Dulse (Palmaria palmata

    Directory of Open Access Journals (Sweden)

    Hanne K. Mæhre

    2016-10-01

    Full Text Available Several common protein extraction protocols have been applied on seaweeds, but extraction yields have been limited. The aims of this study were to further develop and optimize existing extraction protocols and to examine the effect of enzymatic pre-treatment on bioaccessibility and extractability of seaweed proteins. Enzymatic pre-treatment of seaweed samples resulted in a three-fold increase in amino acids available for extraction. Combining enzymatic pre-treatment with alkaline extraction resulted in a 1.6-fold increase in the protein extraction yield compared to a standard alkaline extraction protocol. A simulated in vitro gastrointestinal digestion model showed that enzymatic pre-treatment of seaweed increased the amount of amino acids available for intestinal absorption 3.2-fold. In conclusion, enzymatic pre-treatment of seaweeds is effective for increasing the amount of amino acids available for utilization and may thus be an effective means for increasing the utilization potential of seaweed proteins. However, both the enzymatic pre-treatment protocol and the protein extraction protocol need further optimization in order to obtain optimal cost-benefit and results from the in vitro gastrointestinal digestion model need to be confirmed in clinical models.

  15. Scrapie-associated prion protein accumulates in astrocytes during scrapie infection.

    OpenAIRE

    Diedrich, J F; Bendheim, P E; Kim, Y. S.; Carp, R. I.; Haase, A T

    1991-01-01

    In the course of scrapie, a transmissible spongiform encephalopathy caused by an unconventional agent, a normal cellular protein is converted to an abnormal form that copurifies with infectivity and aggregates to form deposits of amyloid. We have used immunocytochemistry and methods that enhance detection of amyloidogenic proteins to investigate the types of cells in the central nervous system which are involved in the formation of the abnormal scrapie-associated protein. We show that this pr...

  16. Pressurized water extraction of isoflavones by experimental design from soybean flour and Soybean Protein Isolate.

    Science.gov (United States)

    Moras, Benjamin; Rey, Stéphane; Vilarem, Gérard; Pontalier, Pierre-Yves

    2017-01-01

    A Doehlert experimental design was conducted and surface response methodology was used to determine the effect of temperature, contact time and solid liquid ratio on isoflavone extraction from soybean flour or Soybean Protein Isolate in pressurized water system. The optimal conditions conducted gave an extraction yield of 85% from soybean flour. For Soybean Protein Isolate compared to soybean flour, the isoflavone extraction yield is 61%. This difference could be explained by higher aglycon content, while aglycon appears to be the least extracted isoflavone by pressurized water. The solid liquid ratio in the ASE cell was the overriding factor in obtaining high yields with both soybean products, while temperature has less influence. A high temperature causes conversion of the malonyls-glucosides and glucosides isoflavone derivatives into glucosides or aglycons forms. pressurized water extraction showed a high solubilization of protein material up to 95% of inserted Soybean Protein Isolate.

  17. Prevention of free fatty acid-induced lipid accumulation, oxidative stress, and cell death in primary hepatocyte cultures by a Gynostemma pentaphyllum extract.

    Science.gov (United States)

    Müller, C; Gardemann, A; Keilhoff, G; Peter, D; Wiswedel, I; Schild, L

    2012-03-15

    Hepatocytes of a primary cell culture that are exposed to high glucose, insulin, and linoleic (LA) acid concentration respond with lipid accumulation, oxidative stress up to cell death. Such alterations are typically found in patients with non-alcoholic fatty liver disease (NAFLD). We used this cellular model to study the effect of an ethanolic Gynostemma pentaphyllum (GP) extract in NAFLD. When hepatocytes were cultured in the presence of high insulin, glucose, and LA concentration the extract completely protected the cells from cell death. In parallel, the extract prevented accumulation of triglycerides (TGs) and cholesterol as well as oxidative stress. Our data further demonstrate that GP stimulates the production of nitric oxide (NO) in hepatocytes and affects the molecular composition of the mitochondrial phospholipid cardiolipin (CL). We conclude that GP is able to protect hepatocytes from cell death, lipid accumulation, and oxidative stress caused by diabetic-like metabolism and lipotoxicity. Therefore, GP could be beneficial for patients with diabetes mellitus and NAFLD. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. Extraction of Protein-Protein Interaction from Scientific Articles by Predicting Dominant Keywords

    Directory of Open Access Journals (Sweden)

    Shun Koyabu

    2015-01-01

    Full Text Available For the automatic extraction of protein-protein interaction information from scientific articles, a machine learning approach is useful. The classifier is generated from training data represented using several features to decide whether a protein pair in each sentence has an interaction. Such a specific keyword that is directly related to interaction as “bind” or “interact” plays an important role for training classifiers. We call it a dominant keyword that affects the capability of the classifier. Although it is important to identify the dominant keywords, whether a keyword is dominant depends on the context in which it occurs. Therefore, we propose a method for predicting whether a keyword is dominant for each instance. In this method, a keyword that derives imbalanced classification results is tentatively assumed to be a dominant keyword initially. Then the classifiers are separately trained from the instance with and without the assumed dominant keywords. The validity of the assumed dominant keyword is evaluated based on the classification results of the generated classifiers. The assumption is updated by the evaluation result. Repeating this process increases the prediction accuracy of the dominant keyword. Our experimental results using five corpora show the effectiveness of our proposed method with dominant keyword prediction.

  19. Extraction of Protein-Protein Interaction from Scientific Articles by Predicting Dominant Keywords.

    Science.gov (United States)

    Koyabu, Shun; Phan, Thi Thanh Thuy; Ohkawa, Takenao

    2015-01-01

    For the automatic extraction of protein-protein interaction information from scientific articles, a machine learning approach is useful. The classifier is generated from training data represented using several features to decide whether a protein pair in each sentence has an interaction. Such a specific keyword that is directly related to interaction as "bind" or "interact" plays an important role for training classifiers. We call it a dominant keyword that affects the capability of the classifier. Although it is important to identify the dominant keywords, whether a keyword is dominant depends on the context in which it occurs. Therefore, we propose a method for predicting whether a keyword is dominant for each instance. In this method, a keyword that derives imbalanced classification results is tentatively assumed to be a dominant keyword initially. Then the classifiers are separately trained from the instance with and without the assumed dominant keywords. The validity of the assumed dominant keyword is evaluated based on the classification results of the generated classifiers. The assumption is updated by the evaluation result. Repeating this process increases the prediction accuracy of the dominant keyword. Our experimental results using five corpora show the effectiveness of our proposed method with dominant keyword prediction.

  20. Which metaproteome? The impact of protein extraction bias on metaproteomic analyses.

    Science.gov (United States)

    Leary, Dagmar Hajkova; Hervey, W Judson; Deschamps, Jeffrey R; Kusterbeck, Anne W; Vora, Gary J

    2013-01-01

    Culture-independent techniques such as LC-MS/MS-based metaproteomic analyses are being increasingly utilized for the study of microbial composition and function in complex environmental samples. Although several studies have documented the many challenges and sources of bias that must be considered in these types of analyses, none have systematically characterized the effect of protein extraction bias on the biological interpretation of true environmental biofilm metaproteomes. In this study, we compared three protein extraction methods commonly used in the analyses of environmental samples [guanidine hydrochloride (GuHCl), B-PER, sequential citrate-phenol (SCP)] using nano-LC-MS/MS and an environmental marine biofilm to determine the unique biases introduced by each method and their effect on the interpretation of the derived metaproteomes. While the protein extraction efficiencies of the three methods ranged from 2.0 to 4.3%, there was little overlap in the sequence (1.9%), function (8.3% of total assigned protein families) and origin of the identified proteins from each extract. Each extraction method enriched for different protein families (GuHCl - photosynthesis, carbohydrate metabolism; B-PER - membrane transport, oxidative stress; SCP - calcium binding, structural) while 23.7-45.4% of the identified proteins lacked SwissProt annotations. Taken together, the results demonstrated that even the most basic interpretations of this complex microbial assemblage (species composition, ratio of prokaryotic to eukaryotic proteins, predominant functions) varied with little overlap based on the protein extraction method employed. These findings demonstrate the heavy influence of protein extraction on biofilm metaproteomics and provide caveats for the interpretation of such data sets when utilizing single protein extraction methods for the description of complex microbial assemblages.

  1. Reprint of "Which metaproteome? The impact of protein extraction bias on metaproteomic analyses".

    Science.gov (United States)

    Leary, Dagmar Hajkova; Hervey, W Judson; Deschamps, Jeffrey R; Kusterbeck, Anne W; Vora, Gary J

    2014-01-01

    Culture-independent techniques such as LC-MS/MS-based metaproteomic analyses are being increasingly utilized for the study of microbial composition and function in complex environmental samples. Although several studies have documented the many challenges and sources of bias that must be considered in these types of analyses, none have systematically characterized the effect of protein extraction bias on the biological interpretation of true environmental biofilm metaproteomes. In this study, we compared three protein extraction methods commonly used in the analyses of environmental samples [guanidine hydrochloride (GuHCl), B-PER, sequential citrate-phenol (SCP)] using nano-LC-MS/MS and an environmental marine biofilm to determine the unique biases introduced by each method and their effect on the interpretation of the derived metaproteomes. While the protein extraction efficiencies of the three methods ranged from 2.0 to 4.3%, there was little overlap in the sequence (1.9%), function (8.3% of total assigned protein families) and origin of the identified proteins from each extract. Each extraction method enriched for different protein families (GuHCl--photosynthesis, carbohydrate metabolism; B-PER--membrane transport, oxidative stress; SCP--calcium binding, structural) while 23.7-45.4% of the identified proteins lacked SwissProt annotations. Taken together, the results demonstrated that even the most basic interpretations of this complex microbial assemblage (species composition, ratio of prokaryotic to eukaryotic proteins, predominant functions) varied with little overlap based on the protein extraction method employed. These findings demonstrate the heavy influence of protein extraction on biofilm metaproteomics and provide caveats for the interpretation of such data sets when utilizing single protein extraction methods for the description of complex microbial assemblages.

  2. Anticariogenic and Hemolytic Activity of Selected Seed Protein Extracts In vitro conditions.

    Directory of Open Access Journals (Sweden)

    Kalpesh B Ishnava

    2014-10-01

    Full Text Available This study aimed to assess the anticariogenic and hemolytic activity of crude plant seed protein extracts against tooth decaying bacteria.The proteins from seeds of 12 different plants were extracted and used for antimicrobial assay against six different organisms. The extraction was carried out in 10mM of sodium phosphate buffer (pH 7.0. Protein concentrations were determined as described by Bradford method. Anticariogenic activity was studied by agar well diffusion method and Minimum Inhibitory Concentration (MIC was evaluated by the two-fold serial broth dilution method. Hemolytic activity, treatment of proteinase K and Kinetic study in Mimusops elengi crude seed protein extract.The anticariogenic assay demonstrated the activity of Mimusops elengi against Staphylococcus aureus and Streptococcus pyogenes. A minor activity of Glycine wightii against Streptococcus mutans was also found. The protein content of Mimusops elengi seed protein extract was 5.84mg/ml. The MIC values for Staphylococcus aureus and Streptococcus pyogenes against Mimusops elengi seed protein extract were 364.36μg/ml and 182.19μg/ml, respectively. Kinetic study further elucidated the mode of inhibition in the presence of the Mimusops elengi plant seed protein with respect to time. The concentration of crude extract which gave 50% hemolysis compared to Triton X-100 treatment (HC50 value was 1.58 mg/ml; which is more than five times larger than that of the MIC. Treatment with proteinase K of the Mimusops elengi seed protein resulted in absence of the inhibition zone; which clearly indicates that the activity was only due to protein.Our results showed the prominence of Mimusops elengi plant seed protein extract as an effective herbal medication against tooth decaying bacteria.

  3. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts.

    Science.gov (United States)

    Yong, Ann-Li; Ooh, Keng-Fei; Ong, Hean-Chooi; Chai, Tsun-Thai; Wong, Fai-Chu

    2015-11-01

    In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Electrical nerve stimulation and the relief of chronic pain through regulation of the accumulation of synaptic Arc protein.

    Science.gov (United States)

    Liu, Yue-peng; Liu, Su

    2013-08-01

    Electrical nerve stimulation (ENS) is used in clinical settings for the treatment of chronic pain, but the mechanism underlying its effects remains unknown. ENS has been found to mimic neural activity, inducing the accumulation of Arc in synapses. Activity-dependent synaptic accumulation of Arc protein has been shown to reduce synaptic strength by promoting endocytosis of the AMPA receptors in the synaptic membrane. These receptors play a decisive role in central sensitization, which is one of the main mechanisms underlying chronic pain. It is here hypothesized that ENS induces Arc expression in synapses, where Arc promotes endocytosis of membrane AMPARs that are up-regulated during chronic pain. High frequency and high intensity are characteristics of ENS, which may be effective in the treatment of chronic pain. Stimulation-site of ENS may also influence the outcome of ENS.

  5. Analysis of accumulation, extractability, and metabolization of five different phenylarsenic compounds in plants by ion chromatography with mass spectrometric detection and by atomic emission spectroscopy.

    Science.gov (United States)

    Schmidt, Anne-Christine; Kutschera, Kristin; Mattusch, Jürgen; Otto, Matthias

    2008-12-01

    Phenylated arsenic compounds occur as highly toxic contaminants in former military areas where they were formed as degradation products of chemical warfare agents. Some phenylarsenic compounds such as roxarsone and aminophenylarsonic acids were applied as food additive and veterinary drugs in stock-breeding and therefore pose an environmental risk in agricultural used sites. Very few data exist in the literature concerning uptake and effects of phenylarsenic compounds in plants growing on contaminated soils. In this study, the accumulation, extractability, and metabolization of five different phenylarsenic compounds, phenylarsonic acid, p- and o-aminophenylarsonic acid, phenylarsine oxide, and 3-nitro-4-hydroxyphenylarsonic acid called roxarsone, by the terrestrial plant Tropaeolum majus were investigated. Ion chromatography coupled to inductively coupled plasma mass spectrometry was used to differentiate these arsenic compounds, and inductively coupled plasma atomic emission spectroscopy was used for total arsenic quantification. All compounds considered were taken up by the roots and transferred to stalks, leaves, and flowers. The strongest accumulation was observed for unsubstituted phenylarsonic acid followed by its trivalent analogue phenylarsine oxide that was mostly oxidized in soil whereas the amino- or nitro- and hydroxy-substituted phenylarsonic acids were accumulated to a smaller degree. The highest extraction yield of 90% for ground leaf material was achieved by 0.1M phosphate buffer, pH 7.7, in a two-step extraction with a total extraction time of 24h. The extraction of higher amounts of arsenic (50-70% of total arsenic present in leaves depending on arsenic species application) from non-ground intact leaves with deionized water in comparison with the buffer (20-40% of total arsenic) is ascribed to osmotic effects. The arsenic species analysis revealed a cleavage of the amino groups from the phenyl ring for plants treated with aminophenylarsonic acids

  6. Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.; Warner, Marvin G.; Wahl, Karen L.; Hutchison, Janine R.

    2015-08-07

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extraction improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.

  7. Functional Region Identification in Proteins by Accumulative-Quantitative Peptide Mapping Using RP-HPLC-MS

    NARCIS (Netherlands)

    Kuipers, B.J.H.; Bakx, E.J.; Gruppen, H.

    2007-01-01

    A new method was developed to identify regions in proteins from which peptides are derived with specific functional properties. This method is applicable for systems in which peptides of a hydrolyzed protein possess specific functional properties, but are too large to be sequenced directly and/or th

  8. Amino acid accumulation limits the overexpression of proteins in Lactococcus lactis

    NARCIS (Netherlands)

    Marreddy, Ravi K. R.; Geertsma, Eric R.; Permentier, Hjalmar P.; Coelho Pinto, Joao; Kok, Jan; Poolman, Bert

    2010-01-01

    Background: Understanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we emp

  9. PROTEIN EXTRACTION OF MAIZE (ZEA MAYS FOR PROTEOMIC 2 – DE ANALYSIS

    Directory of Open Access Journals (Sweden)

    Edita Gregová

    2014-12-01

    Full Text Available A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. Two different extraction procedures for two-dimensional electrophoresis of plant proteins are compared in this work. Phenol-based extraction methods have been mainly used to extract proteins from different organs or tissues on many species. We wanted to determine which of these protocols was optimal for starch plants in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. The phenol-based protocol was superior to the sodium phosphatase methods, showing larger protein yields and greater spot resolution on 2-D gels.

  10. Independent Research Projects Using Protein Extraction: Affordable Ways to Inquire, Discover & Publish for Undergraduate Students

    Science.gov (United States)

    Pu, Rongsun

    2010-01-01

    This article describes how to use protein extraction, quantification, and analysis in the undergraduate teaching laboratory to engage students in inquiry-based, discovery-driven learning. Detailed instructions for obtaining proteins from animal tissues, using BCA assay to quantify the proteins, and data analysis are provided. The experimental…

  11. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves

    OpenAIRE

    Chen Zhang; Sanders, Johan P. M.; Xiao, Ting T.; Bruins, Marieke E.

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-pr...

  12. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans

    Science.gov (United States)

    Andralojc, Karolina M.; Kelly, Ashley L.; Tanner, Paige C.

    2017-01-01

    Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline. PMID:28182654

  13. ELLI-1, a novel germline protein, modulates RNAi activity and P-granule accumulation in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Karolina M Andralojc

    2017-02-01

    Full Text Available Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3. The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules. ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.

  14. Variations in Carbohydrate and Protein Accumulation among Spikelets at Different Positions Within a Panicle During Rice Grain Filling

    Institute of Scientific and Technical Information of China (English)

    DONG Ming-hui; CHEN Pei-feng; XIE Yu-lin; QIAO Zhong-ying; YANG Jian-chang

    2012-01-01

    The accumulation dynamics of kernel components for spikelets at different positions within a rice panicle were investigated during grain filling to understand the physiological reasons for the variation of grain quality.Two rice cultivars,Yangdao 6 (indica) and Yangjing 9538 (japonica),were field-grown,and the grain filling characters and contents of starch,soluble sugar,and protein of the spikelets at different positions were studied.There were significant differences in matter accumulation among spikelets at different positions during grain filling.The early-flowering spikelets presented dominance over the late-flowering spikelets in initial time and initial rate of accumulation.At the initial and mid filling stages,the contents and the rates of starch and amylose accumulation in spikelets decreased with the flowering sequence,but soluble sugar content (SSC) exhibited the opposite trend.The difference in SSC among the spikelets of Yangjing 9538 was greater than that of Yangdao 6,but amylose content in mature spikelets showed no obvious relationship to their flowering sequence.The crude protein content (CPC) of early-flowering spikelets decreased more rapidly than that of late-flowering ones at the initial filling stage,and CPC in the spikelets on the secondary branch was higher than that on the primary branch,but CPC in early-flowering ones was lower than that in late-flowering across the whole grain filling period.Grain water content (GWC) of early-flowering spikelets decreased more rapidly than that of late-flowering spikelets on the same branch at the initial and mid filling stages,especially for the top grain on each primary branch.The results suggested that poor grain filling of late-flowering spikelets may be attributed to their low biological activity rather than carbohydrate supply limitation.

  15. On-Orbit DNA, RNA, and Protein Extraction Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Genova Engineering proposes to develop and demonstrate a toolset of discrete devices and extraction kits which will leverage existing on-orbit facilities and will...

  16. Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation.

    Science.gov (United States)

    Ehara, Haruhiko; Makino, Marie; Kodama, Koichiro; Konoki, Keiichi; Ito, Takuhiro; Sekine, Shun-ichi; Fukuzawa, Seketsu; Yokoyama, Shigeyuki; Tachibana, Kazuo

    2015-07-06

    Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α-helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.

  17. Current Physical and SDS Extraction Methods Do Not Efficiently Remove Exosporium Proteins from Bacillus anthracis spores

    Science.gov (United States)

    Thompson, Brian M.; Binkley, Jana M.; Stewart, George C.

    2011-01-01

    Biochemical studies of the outermost spore layers of the Bacillus cereus family are hindered by difficulties in efficient dispersal of the external spore layers and difficulties in dissociating protein complexes that comprise the exosporium layer. Detergent and physical methods have been utilized to disrupt the exosporium layer. Herein we compare commonly used SDS extraction buffers used to extract spore proteins and demonstrate the incomplete extractability of the exosporium layer by these methods. Sonication and bead beating methods for exosporium layer removal were also examined. A combination of genetic and physical methods is the most effective for isolating proteins found in the spore exosporium. PMID:21338631

  18. Current physical and SDS extraction methods do not efficiently remove exosporium proteins from Bacillus anthracis spores.

    Science.gov (United States)

    Thompson, Brian M; Binkley, Jana M; Stewart, George C

    2011-05-01

    Biochemical studies of the outermost spore layers of the Bacillus cereus family are hindered by difficulties in efficient dispersal of the external spore layers and difficulties in dissociating protein complexes that comprise the exosporium layer. Detergent and physical methods have been utilized to disrupt the exosporium layer. Herein we compare commonly used SDS extraction buffers used to extract spore proteins and demonstrate the incomplete extractability of the exosporium layer by these methods. Sonication and bead beating methods for exosporium layer removal were also examined. A combination of genetic and physical methods is the most effective for isolating proteins found in the spore exosporium.

  19. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Chapuis, Sophie [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Revers, Frédéric [INRA, Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, 33882 Villenave d’Ornon (France); Ziegler-Graff, Véronique [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Brault, Véronique, E-mail: veronique.brault@colmar.inra.fr [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France)

    2015-12-15

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT{sub Cter}) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT{sub Cter}. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT{sub Cter} in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

  20. Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro.

    Science.gov (United States)

    Jørgensen, Peter; Milkovic, Lidija; Zarkovic, Neven; Waeg, Georg; Rattan, Suresh I S

    2014-02-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

  1. Coronatine Gene Expression In Vitro and In Planta, and Protein Accumulation During Temperature Downshift in Pseudomonas syringae

    Directory of Open Access Journals (Sweden)

    Alexander Schenk

    2009-06-01

    Full Text Available The plant pathogenic bacterium Pseudomonas syringae PG4180 synthesizes high levels of the phytotoxin coronatine (COR at the virulence-promoting temperature of 18 °C, but negligible amounts at 28 °C. Temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of a response regulator, CorR, the histidine protein kinase CorS, and a third component, termed CorP. We analyzed at transcriptional and translational levels the expression of corS and the cma operon involved in COR biosynthesis after a temperature downshift from 28 to 18 °C. Expression of cma was induced within 20 min and increased steadily whereas corS expression was only slightly temperature-dependent. Accumulation of CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis. A transcriptional fusion of the cma promoter to a promoterless egfp gene was used to monitor the cma expression in vitro and in planta. A steady induction of cma::egfp by temperature downshift was observed in both environments. The results indicate that PG4180 responds to a temperature decrease with COR gene expression. However, COR gene expression and protein biosynthesis increased steadily, possibly reflecting adaptation to long-term rather than rapid temperature changes.

  2. Optimal protein extraction methods from diverse sample types for protein profiling by using Two-Dimensional Electrophoresis (2DE).

    Science.gov (United States)

    Tan, A A; Azman, S N; Abdul Rani, N R; Kua, B C; Sasidharan, S; Kiew, L V; Othman, N; Noordin, R; Chen, Y

    2011-12-01

    There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the final results, yet without selectively losing the proteins. To address this, we developed a general method that is suitable for diverse sample types based on phenolchloroform extraction method (represented by TRI reagent). This method was found to yield good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae, Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line, bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit, PRO-PREP™ Protein Extraction Solution and lysis buffer. The most useful protocol allows the extraction and separation of a wide diversity of protein samples that is reproducible among repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the highest protein yield as well as the greatest number of total proteins spots count for all type of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of different extraction methods used for each type of sample.

  3. Determination of the amount of protein and amino acids extracted from the microbial protein (SCP) of lignocellulosic wastes.

    Science.gov (United States)

    Ahmadi, A R; Ghoorchian, H; Hajihosaini, R; Khanifar, J

    2010-04-15

    With the increasing world population, the use of lignocellulosic wastes for production of microbial protein as animal feed becomes a necessity of our time. In order to verify the most productive protein, the amount of protein and amino acid extracted from Single Cell Protein (SCP) needs to be determined by an effective method. In this study Microbial protein was produced by treatment of wheat straw with Pleurotus florida; with heat at 100 degrees C and NaOH 2% as substrate by solid state fermentation. Concentration of protein was 62.8% per 100 g of dried microbial protein. Then the extracted protein hydrolyzed with HCl 6 Normal for 48 h under 110 degrees C temperature condition. Then the amino acids analyzed by using A-200 Amino Nova analyzer. The results of this study indicated that the ratio of essential amino acids to total amino acids was 65.6%. The concentration of essnyial amino acids were: Lysine = 9.5, histidine = 19.8, threonine = 0.6, valine = 6.6, methionine = 2.1, isoleucine = 7.3, leucine = 6.8, phenylalanine = 4.3 and arginine = 8.3 g/100 g of extracted protein that indicated the obtained microbial protein can be a good or suitable substitute in the food program of animal feed.

  4. Use of an enzyme-assisted method to improve protein extraction from olive leaves.

    Science.gov (United States)

    Vergara-Barberán, M; Lerma-García, M J; Herrero-Martínez, J M; Simó-Alfonso, E F

    2015-02-15

    The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55°C for 15min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64mgg(-1)) were analysed and compared by SDS-PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature.

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

    Science.gov (United States)

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

    2013-08-14

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

  6. A green deep eutectic solvent-based aqueous two-phase system for protein extracting

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Kaijia; Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn; Huang, Yanhua; Li, Na; Wen, Qian

    2015-03-15

    Highlights: • A strategy for the protein purification with a deep eutectic solvent(DES)-based aqueous two-phase system. • Choline chloride-glycerin DES was selected as the extraction solvent. • Bovine serum albumin and trypsin were used as the analytes. • Aggregation phenomenon was detected in the mechanism research. - Abstract: As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.

  7. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae)

    Energy Technology Data Exchange (ETDEWEB)

    Chia, Mathias Ahii, E-mail: chia28us@yahoo.com [Department of Botany, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Lombardi, Ana Teresa [Department of Botany, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Graça Gama Melão, Maria da [Department of Hydrobiology, Federal University of São Carlos, Rodovia Washington Luis km 235, São Carlos, SP Cep 13565905 (Brazil); Parrish, Christopher C. [Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, Newfoundland A1C 5S7 (Canada)

    2015-03-15

    Highlights: • Chlorella vulgaris was exposed to Cd under varying N concentrations. • Growth rate and cell density decreased with increasing Cd stress and N limitation. • Dry weight, chlorophyll a, total lipid, carbohydrate and protein were accumulated. • Amino acids like proline and glutamine were accumulated under N and Cd stress. • Changes in amino acid composition are sensitive biomarkers for Cd and N stress. - Abstract: Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10{sup −7} and 2.0 × 10{sup −8} mol L{sup −1} Cd) under varying nitrogen (2.9 × 10{sup −6}, 1.1 × 10{sup −5} and 1.1 × 10{sup −3} mol L{sup −1} N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

  8. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Sato, Atsushi; Matsumura, Rie; Hoshino, Naomi; Tsuzuki, Mikio; Sato, Norihiro

    2014-01-01

    Triacylglycerol (TG) synthesis is induced for energy and carbon storage in algal cells under nitrogen(N)-starved conditions, and helps prevent reactive oxygen species (ROS) production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S)-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P)-starved cells. S- and N- starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase (DGAT) genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N- starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of DGAT genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  9. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Atsushi eSato

    2014-09-01

    Full Text Available Triacylglycerol (TG synthesis is induced for energy and carbon storage in algal cells under nitrogen(N-starved conditions, and helps prevent reactive oxygen species production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P-starved cells. S- and N-starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N-starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of diacylglycerol acyltransferase genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  10. Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance

    Science.gov (United States)

    Sasaki, Yusuke; Ohta, Masayuki; Desai, Dhruv; Figueiredo, Jose-Luiz; Whelan, Mary C.; Sugano, Tomohiro; Yamabi, Masaki; Yano, Wataru; Faits, Tyler; Yabusaki, Katsumi; Zhang, Hengmin; Mlynarchik, Andrew K.; Inoue, Keisuke; Mizuno, Ken; Aikawa, Masanori

    2015-01-01

    Objectives Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. Methodology/Principal Findings Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. Conclusion ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance. PMID:26132105

  11. Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue

    Directory of Open Access Journals (Sweden)

    Jennifer L. Kaplan

    2015-11-01

    Conclusions: This study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21Cip1 expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.

  12. Hydrolysis and loss of extractability of proteins during ripening of iberian ham.

    Science.gov (United States)

    Córdoba, J J; Antequera, T; Ventanas, J; López-Bote, C; García, C; Asensio, M A

    1994-01-01

    To elucidate the extent of the hydrolysis and loss of extractability of protein during the traditional ripening of Iberian ham, the evolution during processing of non-protein nitrogen (NPN) and protein fractions soluble in 0·03 m pH 7·1 phosphate and 1·1 KI + 0·1 m phosphate pH 7·4 buffers and 6 m urea was followed from Semimembranosus and Biceps femoris muscles. The NPN steadily increased during processing, showing maximum intensity at salting and drying. Electrophoretic study of the proteins extracted, and microscopical examination of the pellet obtained after consecutive extractions with the above buffers, revealed that hydrolysis and insolubilization are more intense in myofibrillar than in sarcoplasmic proteins. Protein aggregation involves mainly the myofibrillar fraction, and occurs during the first stage of processing.

  13. Comparison of Two Methods for the Extraction of Fractionated Rice Bran Protein

    Directory of Open Access Journals (Sweden)

    Changyuan Wang

    2014-01-01

    Full Text Available Two different methods for extracting fractionated rice bran protein (FRBP from defatted rice bran were investigated according to the solubility of protein in different extraction solvents. The yields of the obtained proteins and their purity were first compared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, differential scanning calorimetry, protein surface hydrophobicity, and protein secondary molecular structure analyses were subsequently applied to identify and compare the compositional, structural, and functional characteristics of the obtained proteins. The highest yield (13.8%, w/w and purity (45–47% of FRBP products were obtained using 0.4 M NaCl, 80% ethanol, and 0.01 M NaOH as extraction solvents to fractionate albumin, globulin, prolamin, and glutelin. Several good properties were exhibited, including good functionality, specific denaturation temperature, and enthalpy values, for FRBP products prepared by the above method.

  14. Buddleja officinalis Maximowicz Extract Inhibits Lipid Accumulation on Adipocyte Differentiation in 3T3-L1 Cells and High-Fat Mice

    Directory of Open Access Journals (Sweden)

    Jin-Kyu Kim

    2012-07-01

    Full Text Available Obesity is a global health problem. It is also known to be a risk factor for the development of metabolic disorders, type 2 diabetes, systemic hypertension, cardiovascular disease, dyslipidemia, and atherosclerosis. In this study, we elucidated that Buddleja officinalis Maximowicz extract significantly inhibited lipid accumulation during 3T3-L1 adipocyte differentiation. Furthermore, Buddleja officinalis Maximowicz extract reduced the body weight gain induced through feeding a high-fat diet to C57BL/6 mice. The treatment of Buddleja officinalis Maximowicz extract significantly reduced the adipose tissue weight to 2.7/100 g of body weight in high-fat mice. When their adipose tissue morphology was investigated for histochemical staining, the distribution of cell size in the high-fat diet groups was hypertrophied compared with those from Buddleja officinalis Maximowicz extract-treated mice. In addition, in Buddleja officinalis Maximowicz extract-treated mice, a significant reduction of serum triglyceride and T-cholesterol was observed at to 21% and 17%, respectively. The discovery of bioactive compounds from diet or dietary supplementation is one of possible ways to control obesity and to prevent or reduce the risks of various obesity-related diseases. These results support that Buddleja officinalis Maximowicz extract is expected to create the therapeutic interest with respect to the treatment of obesity.

  15. Anti-Inflammatory Effects of Aspalathus linearis and Cyclopia spp. Extracts in a UVB/Keratinocyte (HaCaT Model Utilising Interleukin-1α Accumulation as Biomarker

    Directory of Open Access Journals (Sweden)

    Tandeka Magcwebeba

    2016-10-01

    Full Text Available Ultraviolet B (UVB radiation is one of the major predisposing risk factors of skin cancer. The anticancer and photoprotective effects of unoxidized rooibos (Aspalathus linearis and honeybush (Cyclopia herbal teas, containing high levels of dihydrochalones and xanthones, respectively, have been demonstrated in skin cancer models in vivo. In the current study, the anti-inflammatory effects of methanol and aqueous extracts of these herbal teas were investigated in a UVB/HaCaT keratinocyte model with intracellular interleukin-1α (icIL-1α accumulation as a biomarker. Extracts of green tea (Camellia sinensis served as benchmark. Both extracts of green tea and rooibos, as well as the aqueous extract of C. intermedia, enhanced UVB-induced inhibition of cell viability, proliferation and induction of apoptosis, facilitating the removal of icIL-1α. The underlying mechanisms may involve mitochondrial dysfunction exhibiting pro-oxidant responses via polyphenol-iron interactions. The methanol extracts of honeybush, however, protected against UVB-induced reduction of cell growth parameters, presumably via antioxidant mechanisms that prevented the removal of highly inflamed icIL-1α-containing keratinocytes via apoptosis. The dual antioxidant and/or pro-oxidant role of the polyphenolic herbal tea constituents should be considered in developing preventive strategies against UVB-induced skin carcinogenesis. The indirect removal of UVB damaged keratinocytes by herbal tea extracts via apoptosis may find application in the prevention of photo-induced inflammation.

  16. The effect of temperature and bacterial growth phase on protein extraction by means of electroporation.

    Science.gov (United States)

    Haberl-Meglič, Saša; Levičnik, Eva; Luengo, Elisa; Raso, Javier; Miklavčič, Damijan

    2016-12-01

    Different chemical and physical methods are used for extraction of proteins from bacteria, which are used in variety of fields. But on a large scale, many methods have severe drawbacks. Recently, extraction by means of electroporation showed a great potential to quickly obtain proteins from bacteria. Since many parameters are affecting the yield of extracted proteins, our aim was to investigate the effect of temperature and bacterial growth phase on the yield of extracted proteins. At the same time bacterial viability was tested. Our results showed that the temperature has a great effect on protein extraction, the best temperature post treatment being 4°C. No effect on bacterial viability was observed for all temperatures tested. Also bacterial growth phase did not affect the yield of extracted proteins or bacterial viability. Nevertheless, further experiments may need to be performed to confirm this observation, since only one incubation temperature (4°C) and one incubation time before and after electroporation (0.5 and 1h) were tested for bacterial growth phase. Based on our results we conclude that temperature is a key element for bacterial membrane to stay in a permeabilized state, so more proteins flow out of bacteria into surrounding media.

  17. Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification.

    Science.gov (United States)

    Li, Shi-Yan; Du, Min; Dolence, E Kurt; Fang, Cindy X; Mayer, Gabriele E; Ceylan-Isik, Asli F; LaCour, Karissa H; Yang, Xiaoping; Wilbert, Christopher J; Sreejayan, Nair; Ren, Jun

    2005-04-01

    Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24-26-month) mice using a MyoCam system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, +/- dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was approximately 2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4-7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs.

  18. Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue.

    Science.gov (United States)

    Griffiths, Leigh G; Choe, Leila; Lee, Kelvin H; Reardon, Kenneth F; Orton, E Christopher

    2008-11-01

    Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.

  19. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    Science.gov (United States)

    Li, Shikuo; Shen, Yuhua; Xie, Anjian; Yu, Xuerong; Zhang, Xiuzhen; Yang, Liangbao; Li, Chuanhao

    2007-10-01

    We describe the formation of amorphous selenium (α-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO32-) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO32- ions to Se0, but also controls the nucleation and growth of Se0, and even participates in the formation of α-Se/protein composites. The size and shell thickness of the α-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO32- ions by Capsicum annuum L extract.

  20. Inhibition of thermal induced protein denaturation of extract/fractions of Withania somnifera and isolated withanolides.

    Science.gov (United States)

    Khan, Murad Ali; Khan, Haroon; Rauf, Abdul; Ben Hadda, Taibi

    2015-01-01

    This study describes the in vitro inhibition of protein denaturation of extract/fractions of Withania somnifera and isolated withanolides including 20β hydroxy-1-oxo(22R)-witha-2,5,24 trienolide (1), (20R,22R-14α,20α)-dihydroxy-1-oxowitha-2,5,16,24 tetraenolide (2). The results showed that the extract/fractions of the plant evoked profound inhibitory effect on thermal-induced protein denaturation. The chloroform fraction caused the most dominant attenuation of 68% at 500 μg/mL. The bioactivity-guided isolation from chloroform fraction led to the isolation of compounds 1 and 2 that showed profound protein inhibition with 78.05% and 80.43% effect at 500 μg/mL and thus strongly complimented the activity of extract/fractions. In conclusion, extract/fractions of W. somnifera possessed strong inhibition of protein denaturation that can be attributed to these isolated withanolides.

  1. Extraction of Jatropha curcas proteins and application in polyketone-based wood adhesives

    NARCIS (Netherlands)

    Hamarneh, A. I.; Heeres, H. J.; Broekhuis, A. A.; Picchioni, F.

    2010-01-01

    Jatropha proteins were successfully extracted from the corresponding seeds using the principle of isoelectric precipitation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), elemental analysis and Fourier transform infrared spectroscopy (FTIR) were used to analyze the obtained p

  2. Comparative proteomic analysis using 2DE-LC-MS/MS reveals the mechanism of Fuzhuan brick tea extract against hepatic fat accumulation in rats with nonalcoholic fatty liver disease.

    Science.gov (United States)

    Liu, Zhonghua; Lin, Yong; Zhang, Sheng; Wang, Die; Liang, Qionglin; Luo, Guoan

    2015-09-01

    Fuzhuan brick tea has received increasing attention in recent years owing to its benefits for nonalcoholic fatty liver disease (NAFLD) and associated metabolic syndrome. For exploring the ameliorative mechanism, the liver proteomes from three groups of rats fed either a normal control diet (NCD), a high fat diet (HFD), or a HFD supplemented with high-dose Fuzhuan brick tea extract (FTE) (HFD + HFTE) were comprehensively compared by quantitative proteomics using 2DE-LC-MS/MS. This is the first study of the effects of tea aqueous extract on the liver proteome of rats suffering from metabolic syndrome. The results showed that 57 proteins displayed more than 1.5-fold differences in at least one of two comparisons of HFD versus NCD and HFD versus HFD + HFTE due to HFD feeding and FTE treatment, respectively. Of them, over 75% of proteins exhibited a similar tendency of expression in the two comparisons, meaning FTE was able to correct HFD effects on rat livers. By function analyses, an extensive list of proteins was involved in sugar and lipid metabolism. Compared with HFD-fed rats, the reduced lipogenesis and enhanced β-oxidation, tricarboxylic acid cycle and respiratory chain in HFD + HFTE-fed rats, which mainly contributed to ameliorate hepatic fat accumulation and associated NAFLD. Additionally, some putative drug targets were also revealed such as COX2, PGAM1, ACACB, FAS, and ECHS1.

  3. Effects of Microwave and Ultrasound Assisted Extraction on the Recovery of Soy Proteins for Soy Allergen Detection.

    Science.gov (United States)

    Amponsah, Amma; Nayak, Balunkeswar

    2016-10-14

    The extraction of soy proteins for soy allergen detections is conventionally achieved with PBS buffer for at least 2 h at room temperature or 4 °C. This method has been reported to be inefficient due to time consumption and inadequate protein extraction resulting in false negative allergen detection and mislabeling of foods containing allergenic proteins. This study investigated the application of microwave (MAE) and ultrasound assisted extraction (UAE) techniques to extract and improve recovery of allergens from various soy matrices. Soy proteins were extracted from raw soy flour, soy protein isolate (SPI) and soy milk using MAE at 60, 70, and 100 °C for 5 and 10 min and UAE at 4 and 23 °C for extraction times of 1, 5, and 10 min with PBS, Laemmli and urea buffers. Extracts were analyzed for total proteins, protein profile, and antibody-based detection (ELISA) of soy proteins. Conventional extraction with each of the buffers was used as controls. Overall, proteins recovered from MAE and UAE samples were higher than recoveries from the controls in all soy matrices. Under all extraction conditions, Laemmli and urea buffer recovered more proteins than PBS. Electrophoresis analysis of protein showed bands around 75, 50, and 33 kDa indicating the presence of soy allergenic proteins β-conglycinin and glycinin, in all samples. Using sandwich ELISA, control and UAE extracts resulted in high soy protein detection but this reduced in MAE extracts.

  4. Evaluation of extraction procedures for 2-DE analysis of aphid proteins.

    Science.gov (United States)

    Yiou, Pan; Shaoli, An; Kebin, Li; Tao, Wang; Kui, Fang; Hua, Zhang; Yu, Sun; Xun, Yang; Jinghui, Xi

    2013-02-01

    Protein sample preparation is a crucial step in a 2-DE proteomics approach. In order to establish a routine protocol for the application of proteomics analysis to aphids, this study focuses on the specific protein extraction problems in insect tissues and evaluates four methods to bypass them. The approaches of phenol extraction methanol/ammonium acetate precipitation (PA), TCA/acetone precipitation, PEG precipitation, and no precipitation were evaluated for proteins isolation and purification from apterous adult aphids, Sitobion avenae. For 2-DE, the PA protocol was optimal, resulting in good IEF and clear spots. PA method yielded the greatest amount of protein and displayed most protein spots in 2-DE gels, as compared with the TCA/acetone precipitation, PEG precipitation and no precipitation protocols. Analysis of protein yield, image quality and spot numbers demonstrate that the TCA/acetone precipitation protocol is a reproducible and reliable method for extracting proteins from aphids. The PEG precipitation approach is a newly developed protein extraction protocol for aphids, from which more unique protein spots can be detected, especially for detection of acid proteins. These protocols are expected to be applicable to other insects or could be of interest to laboratories involved in insect proteomics, despite the amounts and types of interfering compounds vary considerably in different insects.

  5. Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves.

    Science.gov (United States)

    Zhang, Meide; Shen, Shihua

    2013-07-01

    Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods-trichloroacetic acid/acetone, Mg/NP-40, and phenol/ammonium acetate-were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low-abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP-40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.

  6. Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

    Science.gov (United States)

    Al-Obaidi, Jameel R; Saidi, Noor Baity; Usuldin, Siti Rokhiyah Ahmad; Hussin, Siti Nahdatul Isnaini Said; Yusoff, Noornabeela Md; Idris, Abu Seman

    2016-04-01

    Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.

  7. Comparison of Protein Extracts from Various Unicellular Green Sources

    NARCIS (Netherlands)

    Teuling, Emma; Wierenga, Peter A.; Schrama, Johan W.; Gruppen, Harry

    2017-01-01

    Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation

  8. Extraction and characterisation of protein fractions from five insect species

    NARCIS (Netherlands)

    Yi, L.; Lakemond, C.M.M.; Sagis, L.M.C.; Eisner-Schadler, V.R.; Huis, van A.; Boekel, van M.A.J.S.

    2013-01-01

    Tenebrio molitor, Zophobas morio, Alphitobius diaperinus, Acheta domesticus and Blaptica dubia were evaluated for their potential as a future protein source. Crude protein content ranged from 19% to 22% (Dumas analysis). Essential amino acid levels in all insect species were comparable with soybean

  9. Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein.

    Science.gov (United States)

    An, Bang; Li, Boqiang; Qin, Guozheng; Tian, Shiping

    2012-08-01

    In this article, we investigated the effect of exogenous calcium on improving viability of Debaryomyces hansenii and Pichia membranaefaciens under heat stress, and evaluated the role of calcium in reducing oxidant damage of proteins in the yeast cells. The results indicated that high concentration of exogenous calcium in culture medium was beneficial for enhancing the tolerance of the biocontrol yeasts to heat stress. The possible mechanism of calcium improving the viability of yeasts was attributed to enhancement of antioxidant enzyme activities, decrease in ROS accumulation and reduction of oxidative damage of intracellular protein in yeast cells under heat stress. D. hansenii is more sensitive to calcium as compared to P. membranaefaciens. Our results suggest that application of exogenous calcium combined with biocontrol yeasts is a practical approach for the control of postharvest disease in fruit.

  10. Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, T.; Kobayashi, T.; Shinnoh, N.; Goto, I. (Kyushu Univ., Fukuoka (Japan))

    1990-10-01

    In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous (32P)ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using (3H)methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the (3H) methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.

  11. Enzymatic extractability of soybean meal proteins and carbohydrates : heat and humidity effects

    NARCIS (Netherlands)

    Fischer, M.; Kofod, L.V.; Schols, H.A.; Piersma, S.R.; Gruppen, H.; Voragen, A.G.J.

    2001-01-01

    To study the incomplete enzymatic extractability of proteins and carbohydrates of thermally treated soybean meals, one unheated and three heat-treated soybean meals were produced. To obtain truly enzyme-resistant material, the meals were extracted by a repeated hydrolysis procedure using excessive

  12. In vivo accumulation of Helicobacter pylori products, NOD1, ubiquitinated proteins and proteasome in a novel cytoplasmic structure.

    Directory of Open Access Journals (Sweden)

    Vittorio Necchi

    Full Text Available Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer, are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance.

  13. In vivo accumulation of Helicobacter pylori products, NOD1, ubiquitinated proteins and proteasome in a novel cytoplasmic structure.

    Science.gov (United States)

    Necchi, Vittorio; Sommi, Patrizia; Ricci, Vittorio; Solcia, Enrico

    2010-03-16

    Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS) we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance.

  14. In Vivo Accumulation of Helicobacter pylori Products, NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure

    Science.gov (United States)

    Necchi, Vittorio; Sommi, Patrizia; Ricci, Vittorio; Solcia, Enrico

    2010-01-01

    Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS) we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance. PMID:20300534

  15. Cardiac expression of microsomal triglyceride transfer protein is increased in obesity and serves to attenuate cardiac triglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Emil D Bartels

    Full Text Available Obesity causes lipid accumulation in the heart and may lead to lipotoxic heart disease. Traditionally, the size of the cardiac triglyceride pool is thought to reflect the balance between uptake and beta-oxidation of fatty acids. However, triglycerides can also be exported from cardiomyocytes via secretion of apolipoproteinB-containing (apoB lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP; the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism remain unknown, we investigated how cardiac lipoprotein synthesis affects cardiac expression of triglyceride metabolism-controlling genes, insulin sensitivity, and function in obese mice. Heart-specific ablation of MTP-A in mice using Cre-loxP technology impaired upregulation of MTP expression in response to increased fatty acid availability during fasting and fat feeding. This resulted in cardiac triglyceride accumulation but unaffected cardiac insulin-stimulated glucose uptake. Long-term fat-feeding of male C57Bl/6 mice increased cardiac triglycerides, induced cardiac expression of triglyceride metabolism-controlling genes and attenuated heart function. Abolishing cardiac triglyceride accumulation in fat-fed mice by overexpression of an apoB transgene in the heart prevented the induction of triglyceride metabolism-controlling genes and improved heart function. The results suggest that in obesity, the physiological increase of cardiac MTP expression serves to attenuate cardiac triglyceride accumulation albeit without major effects on cardiac insulin sensitivity. Nevertheless, the data suggest that genetically increased lipoprotein secretion prevents development of obesity-induced lipotoxic heart disease.

  16. Cardiac Expression of Microsomal Triglyceride Transfer Protein Is Increased in Obesity and Serves to Attenuate Cardiac Triglyceride Accumulation

    Science.gov (United States)

    Bartels, Emil D.; Nielsen, Jan M.; Hellgren, Lars I.; Ploug, Thorkil; Nielsen, Lars B.

    2009-01-01

    Obesity causes lipid accumulation in the heart and may lead to lipotoxic heart disease. Traditionally, the size of the cardiac triglyceride pool is thought to reflect the balance between uptake and β-oxidation of fatty acids. However, triglycerides can also be exported from cardiomyocytes via secretion of apolipoproteinB-containing (apoB) lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP); the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism remain unknown, we investigated how cardiac lipoprotein synthesis affects cardiac expression of triglyceride metabolism-controlling genes, insulin sensitivity, and function in obese mice. Heart-specific ablation of MTP-A in mice using Cre-loxP technology impaired upregulation of MTP expression in response to increased fatty acid availability during fasting and fat feeding. This resulted in cardiac triglyceride accumulation but unaffected cardiac insulin-stimulated glucose uptake. Long-term fat-feeding of male C57Bl/6 mice increased cardiac triglycerides, induced cardiac expression of triglyceride metabolism-controlling genes and attenuated heart function. Abolishing cardiac triglyceride accumulation in fat-fed mice by overexpression of an apoB transgene in the heart prevented the induction of triglyceride metabolism-controlling genes and improved heart function. The results suggest that in obesity, the physiological increase of cardiac MTP expression serves to attenuate cardiac triglyceride accumulation albeit without major effects on cardiac insulin sensitivity. Nevertheless, the data suggest that genetically increased lipoprotein secretion prevents development of obesity-induced lipotoxic heart disease. PMID:19390571

  17. A Cohort Study of p53 Mutations and Protein Accumulation in Benign Breast Tissue and Subsequent Breast Cancer Risk.

    Science.gov (United States)

    Kabat, Geoffrey C; Kandel, Rita A; Glass, Andrew G; Jones, Joan G; Olson, Neal; Duggan, Catherine; Ginsberg, Mindy; Negassa, Abdissa; Rohan, Thomas E

    2011-01-01

    Mutations in the p53 tumor suppressor gene and accumulation of its protein in breast tissue are thought to play a role in breast carcinogenesis. However, few studies have prospectively investigated the association of p53 immunopositivity and/or p53 alterations in women with benign breast disease in relation to the subsequent risk of invasive breast cancer. We carried out a case-control study nested within a large cohort of women biopsied for benign breast disease in order to address this question. After exclusions, 491 breast cancer cases and 471 controls were available for analysis. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (95% CI). Neither p53 immunopositivity nor genetic alterations in p53 (either missense mutations or polymorphisms) was associated with altered risk of subsequent breast cancer. However, the combination of both p53 immunopositivity and any p53 nucleotide change was associated with an approximate 5-fold nonsignificant increase in risk (adjusted OR 4.79, 95% CI 0.28-82.31) but the confidence intervals were extremely wide. Our findings raise the possibility that the combination of p53 protein accumulation and the presence of genetic alterations may identify a group at increased risk of breast cancer.

  18. A Cohort Study of p53 Mutations and Protein Accumulation in Benign Breast Tissue and Subsequent Breast Cancer Risk

    Directory of Open Access Journals (Sweden)

    Geoffrey C. Kabat

    2011-01-01

    Full Text Available Mutations in the p53 tumor suppressor gene and accumulation of its protein in breast tissue are thought to play a role in breast carcinogenesis. However, few studies have prospectively investigated the association of p53 immunopositivity and/or p53 alterations in women with benign breast disease in relation to the subsequent risk of invasive breast cancer. We carried out a case-control study nested within a large cohort of women biopsied for benign breast disease in order to address this question. After exclusions, 491 breast cancer cases and 471 controls were available for analysis. Unconditional logistic regression was used to estimate odds ratios (OR and 95% confidence intervals (95% CI. Neither p53 immunopositivity nor genetic alterations in p53 (either missense mutations or polymorphisms was associated with altered risk of subsequent breast cancer. However, the combination of both p53 immunopositivity and any p53 nucleotide change was associated with an approximate 5-fold nonsignificant increase in risk (adjusted OR 4.79, 95% CI 0.28–82.31 but the confidence intervals were extremely wide. Our findings raise the possibility that the combination of p53 protein accumulation and the presence of genetic alterations may identify a group at increased risk of breast cancer.

  19. Small deletions in the potato leafroll virus readthrough protein affect particle morphology, aphid transmission, virus movement and accumulation.

    Science.gov (United States)

    Peter, Kari A; Liang, Delin; Palukaitis, Peter; Gray, Stewart M

    2008-08-01

    Potato leafroll virus (PLRV) capsid comprises 180 coat protein (CP) subunits, with some percentage containing a readthrough domain (RTD) extension located on the particle's surface. The RTD N terminus is highly conserved in luteovirids and this study sought to identify biologically active sites within this region of the PLRV RTD. Fourteen three-amino-acid-deletion mutants were generated from a cloned infectious PLRV cDNA and delivered to plants by Agrobacterium inoculations. All mutant viruses accumulated locally in infiltrated tissues and expressed the readthrough protein (RTP) containing the CP and RTD sequences in plant tissues; however, when purified, only three mutant viruses incorporated the RTP into the virion. None of the mutant viruses were aphid transmissible, but the viruses persisted in aphids for a period sufficient to allow for virus transmission. Several mutant viruses were examined further for systemic infection in four host species. All mutant viruses, regardless of RTP incorporation, moved systemically in each host, although they accumulated at different rates in systemically infected tissues. The biological properties of the RTP are sensitive to modifications in both the RTD conserved and variable regions.

  20. Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas

    Energy Technology Data Exchange (ETDEWEB)

    Warchalowska-Sliwa, E. [Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Slawkowska 17, 31-016 Cracow (Poland)]. E-mail: warchalowska@isez.pan.krakow.pl; Niklinska, M. [Department of Ecotoxicology, Institute of Environmental Studies, Jagiellonian University, Gronostajowa 3, 30-387 Cracow (Poland); Goerlich, A. [Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow (Poland); Michailova, P. [Institute of Zoology, Bulgarian Academy of Sciences, 1 Tzar Osvoboditiel Boulevard, Sofia 1000 (Bulgaria); Pyza, E. [Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow (Poland)

    2005-01-01

    The orthopteran insect Tetrix tenuicornis, collected from polluted and unpolluted areas, was used to study heavy metal accumulation and its impact on stress protein levels and on changes in the number and morphology of chromosomes in mitotic and meiotic cells. During two consecutive years, insects were collected from polluted areas of zinc-lead mine spoils near Boleslaw (Poland) and from unpolluted areas near Busko and Staszow (Poland). T. tenuicornis from the polluted area showed 1.5, 4.03, 4.32 and 41.73 times higher concentrations of copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd), respectively, than insects of the same species collected from unpolluted areas. Insects exposed to heavy metals showed only small changes, and rather a decrease in the concentration of constitutive and inducible heat shock proteins Hsp70, the level of which increases under stress conditions. A cytogenetic study of T. tenuicornis revealed intra-population anomalies in chromosome number and morphology in mitotic and meiotic cells and the presence of an additional B chromosome in germinal cells. In 50% of females collected from polluted areas, mosaic oogonial mitotic chromosome sets and diploid, hypo- or hypertetraploid, tetraploid, and octoploid chromosome numbers were detected. In turn, 14.6% of males showed a heterozygous deficiency of chromatin in L{sub 2} and M{sub 3} bivalents in addition to the presence of B chromosomes. - Metals accumulation caused genotoxicity in insects.

  1. Induced systemic resistance in radish is not associated with accumulation of pathogenesis-related proteins

    NARCIS (Netherlands)

    Hoffland, E.; Pieterse, C.M.J.; Bik, L.; Pelt, J.A. van den

    1995-01-01

    The non-pathogenic Pseudomonas fluorescens strain WCS417r has been shown to induce systemic resistance in radish against Fusarium oxysporum f.sp. raphani. In this paper we investigate the involvement of pathogenesis-related (PR) proteins in this Pseudomonas-induced resistance. For comparison, salicy

  2. Proteomic analysis of Pigeonpea (cajanus cajan) seeds reveals the accumulation of numerous stress-related proteins

    Science.gov (United States)

    Pigeonpea is one of the major sources of dietary protein for more than a billion people living in South Asia. This hardy legume is often grown in low-input and risk-prone marginal environments. Considerable research effort has been devoted by a global research consortium to develop genomic resources...

  3. Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

    Science.gov (United States)

    Bermejo, B; Remacha, M; Ortiz-Reyes, B; Santos, C; Ballesta, J P

    1994-02-11

    Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

  4. Influence of berry ripeness on accumulation, composition and extractability of skin and seed flavonoids in cv. Sangiovese (Vitis vinifera L.).

    Science.gov (United States)

    Allegro, Gianluca; Pastore, Chiara; Valentini, Gabriele; Muzzi, Enrico; Filippetti, Ilaria

    2016-10-01

    The anthocyanin and tannin concentration and composition of Vitis vinifera L. cv. Sangiovese berries were investigated from post-veraison to harvest. Exhaustive extraction with methanol and acetone was performed to determine the total flavonoid concentration, while a model hydroalcoholic solution was used to prepare extracts representing the winemaking process. The aim of this study was to improve the knowledge of the phenolic maturity of Sangiovese grape. The total anthocyanin concentration increased during ripening, but the quantity of extractable anthocyanins increased more rapidly than the total. The total skin tannin concentration declined from post-veraison to harvest, whereas the extractable portion increased, with little difference in the composition of the fractions. Both the total and extractable seed tannin concentration diminished rapidly just after veraison, and only small fluctuations were detected until harvest. These results indicate that the extractability of anthocyanins and skin tannins increases during ripening, whereas there is no clear trend for seed tannins during the same period. This is the first survey to study the behavior of phenolic compounds during different steps of ripening of Sangiovese grape. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  5. MIPCE: An MI-based protein complex extraction technique

    Indian Academy of Sciences (India)

    Priyakshi Mahanta; Dhruba K R Bhattacharyya; Ashish Ghosh

    2015-10-01

    Protein–protein interaction (PPI) networks are believed to be important sources of information related to biological processes and complex metabolic functions of the cell. Identifying protein complexes is of great importance for understanding cellular organization and functions of organisms. In this work, a method is proposed, referred to as MIPCE, to find protein complexes in a PPI network based on mutual information. MIPCE has been biologically validated by GO-based score and satisfactory results have been obtained. We have also compared our method with some well-known methods and obtained better results in terms of various parameters such as precession, recall and F-measure.

  6. The Extract of Litsea japonica Reduced the Development of Diabetic Nephropathy via the Inhibition of Advanced Glycation End Products Accumulation in db/db Mice

    Directory of Open Access Journals (Sweden)

    Eunjin Sohn

    2013-01-01

    Full Text Available Increasing evidence indicates that advanced glycation end products (AGEs contribute to the pathogenesis of diabetic nephropathy. The aim of this study was to investigate the protective effect of L. japonica extract (LJE against renal damage in the db/db mouse. LJE (100 or 250 mg/kg per day was given to diabetic mice for 12 weeks. Body weight, blood glucose levels, glycated hemoglobin (HbA1c levels, and proteinuria were examined. In in vitro assay of the inhibition of AGE formation, immunohistochemical analysis of podocyte loss and AGE accumulations were performed. In 20-week-old db/db mice, severe hyperglycemia developed, and proteinuria was significantly increased. Diabetes induced markedly morphological alterations to the renal glomerular cells. AGE accumulations and podocyte loss were detected in renal glomeruli. LJE treatment significantly reduced proteinuria and AGE accumulations in diabetic mice. Moreover, the loss of nephrin, an important slit diaphragm component in the kidneys, was restored by LJE treatment. Our studies suggest that LJE might be beneficial for the treatment of diabetic nephropathy. The ability of LJE to attenuate proteinuria and podocyte dysfunction may be mediated by the inhibition of AGE accumulation in the context of diabetic nephropathy in db/db mice.

  7. Alcohol-extracted, but not intact, dietary soy protein lowers lipoprotein(a) markedly

    DEFF Research Database (Denmark)

    Meinertz, Hans; Nilausen, Karin; Hilden, Jørgen

    2002-01-01

    , liquid-formula diets containing casein, soy protein, or alcohol-extracted soy protein. Dietary periods of 32 days were separated by washout periods on self-selected diets. Fasting lipid and Lp(a) levels were measured throughout. Median Lp(a) concentration was >2-fold greater after 28 to 32 days on a soy...... protein diet than after an extracted soy protein diet (Pextracted soy protein diets were virtually identical. Women and men responded similarly. When the switch was made from a self-selected to a soy protein diet, median Lp(a) concentration increased 16......% after 1 week (Pextracted soy protein and casein diets never exhibited increased median Lp(a) levels, and after 28 to 32 days, these levels were decreased >60% below baseline (P

  8. PENGHAMBATAN OKSIDASI LDL DAN AKUMULASI KOLESTEROL PADA MAKROFAG OLEH EKSTRAK TEMULAWAK (Curcuma xanthorriza Roxb [The Inhibition of Low Density Lipoprotein Oxidation and Cholesterol Accumulation on the Macrophage by Temulawak Extract

    Directory of Open Access Journals (Sweden)

    Aisyah Tri Septiana1

    2006-12-01

    Full Text Available Coronary heart disease is caused among others by atherosclerosis, which is the result of oxidized low density lipoprotein (LDL and cholesterol accumulation on macrophage, and which is inhibited by temulawak (Curcuma xanthorriza Roxb extract. The objective of this study was to find out the kinds and consentration of temulawak extract which could inhibit LDL oxidation, and to find out the effect of temulawak extract on the accumulation of cholesterol on macrophage. Temulawak was extracted by water, ethanol, aceton and dichlorometane. Inhibition of LDL oxidation was found out by measuring the level of malonaldehyde content of oxidized LDL-CuSO4 which was supplemented with water extract, ethanol extract, aceton extract and dichlorometane extract. of temulawak at concentrations of 43 g, 430 g, and 4300 g per ml of LDL. The percentage of malonaldehyde reduction due to supplementation with water extract, ethanol extract, acetone extract and dichloromethane extract was 44.27; 47.68; 51.83 and 61.2 respectively. The inhibition of LDL oxidation by temulawak extract depends on its concentration. The percentage of malonaldehyde reduction due to supplementation with temulawak extract of 43 µg, 430 µg, and 4300 µg per ml of LDL was 43.63; 56.72; and 53.89.. Concentration of temulawak extract resulting in the highest inhibition of LDL oxidation was 430 µg/ml LDL. Temulawak extract tends to inhibit cholesterol accumulation on macrophage. There is a relationship between the inhibition of cholesterol accumulation on the macrophage and the inhibition of LDL oxidation by temulawak extract

  9. Evaluation of four protein extraction methods for proteomic analysis of mango peel.

    Science.gov (United States)

    Liao, D J; Lu, X P; Chen, H S; Lu, Y; Mo, Z Y

    2016-08-30

    The peel of mango (Mangifera indica L.) is a special plant tissue that contains many compounds that interfere with protein extraction. A successful separation with Two-dimensional electrophoresis (2-DE) is the key step for proteomic analysis. To evaluate the efficiencies of mango peel protein extraction for 2-DE, four extraction methods were tested: 1) 2-D clean-up kit, 2) trichloroacetic acid/acetone precipitation, 3) phenol extraction, 4) phenol with methanol/ammonium acetate precipitation. The results showed that the phenol with methanol/ammonium acetate precipitation produced the best quality protein extraction and separation. Proteins were separated in 30-70 and >70 kDa ranges better than with the other methods. Acidic proteins had better resolution with fewer horizontal and vertical streaks. Sixteen proteins were identified by maxtrix-assisted laser desorption/ ionisation time-of-flight tandem mass spectrometry (MALDI-TOF/ TOF-MS/MS). The result demonstrated that each of these four methods can be used to prepare mango peel proteins. The phenol with methanol/ ammonium acetate precipitation was the best choice for proteomic analysis of mango peel.

  10. Accumulation of guaiacol glycoconjugates in fruit, leaves and shoots of Vitis vinifera cv. Monastrell following foliar applications of guaiacol or oak extract to grapevines.

    Science.gov (United States)

    Pardo-Garcia, Ana I; Wilkinson, Kerry L; Culbert, Julie A; Lloyd, Natoiya D R; Alonso, Gonzalo L; Salinas, M Rosario

    2017-02-15

    Previous studies have shown that volatile compounds present within a vineyard during the growing season can be absorbed by grapevines, assimilated within grapes, and then released during fermentation to influence the final aroma of wine. For example, the accumulation of volatile phenols in glycoconjugate forms following grapevine exposure to bushfire smoke, and their subsequent release during winemaking. This study investigated the accumulation of guaiacol glycoconjugates in the fruit, shoots and leaves of Monastrell grapevines following foliar applications (at veraison) of either an aqueous solution of guaiacol or an aqueous oak extract. Fruit, shoot and leaf samples were then collected at 3 time points between veraison and maturity, and analysed by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, to quantify guaiacol and its glycoconjugates, respectively. Guaiacol glycoconjugates were observed in fruit and leaves in particular, demonstrating glycosylation occurred after grapevine treatment; however, different glycoconjugate profiles were apparent.

  11. Cardiac remodeling associated with protein increase and lipid accumulation in early-stage chronic kidney disease in rats.

    Science.gov (United States)

    Kuwahara, Mieko; Bannai, Kenji; Segawa, Hiroko; Miyamoto, Ken-ichi; Yamato, Hideyuki

    2014-09-01

    Chronic kidney disease (CKD) is associated with increased risks of cardiovascular morbidity and mortality. Cardiac remodeling including myocardial fibrosis and hypertrophy is frequently observed in CKD patients. In this study, we investigate the mechanism involved in cardiac hypertrophy associated with CKD using a rat model, by morphological and chemical component changes of the hypertrophic and non-hypertrophic hearts. Sprague-Dawley rats were 4/5 nephrectomized (Nx) at 11 weeks of age and assigned to no treatment and treatment with AST-120, which was reported to affect the cardiac damage, at 18 weeks of age. At 26 weeks of age, the rats were euthanized under anesthesia, and biochemical tests as well as analysis of cardiac condition were performed by histological and spectrophotometric methods. Cardiac hypertrophy and CKD were observed in 4/5 Nx rats even though vascular calcification and myocardial fibrosis were not detected. The increasing myocardial protein was confirmed in hypertrophic hearts by infrared spectroscopy. The absorption of amide I and other protein bands in hypertrophic hearts increased at the same position as in normal cardiac absorption. Infrared spectra also showed that lipid accumulation was also detected in hypertrophic heart. Conversely, the absorptions of protein were obviously reduced in the myocardium of non-hypertrophic heart with CKD compared to that of hypertrophic heart. The lipid associated absorption was also decreased in non-hypertrophic heart. Our results suggest that cardiac remodeling associated with relatively early-stage CKD may be suppressed by reducing increased myocardial protein and ameliorating cardiac lipid load.

  12. Use of carbonate extraction in analyzing moderately hydrophobic transmembrane proteins in the mitochondrial inner membrane.

    Science.gov (United States)

    Kim, Hayoung; Botelho, Salomé Calado; Park, Kwangjin; Kim, Hyun

    2015-12-01

    Resistance to sodium carbonate extraction is regarded as a canonical way to distinguish integral membrane proteins (MPs) from other membrane-associated proteins. However, it has been observed that carbonate extraction releases some mitochondrial integral MPs. Here, by analyzing both artificially designed and native mitochondrial inner MPs containing transmembrane domains (TMDs) of different hydrophobicities, we show that carbonate treatment can release moderately hydrophobic TMDs from the mitochondrial inner membrane. These results suggest that resistance and sensitivity to carbonate extraction may be interpreted with caution when analyzing the nature of mitochondrial inner MPs.

  13. Evaluation of effect of alcoholic extract of heartwood of Pterocarpus marsupium on in vitro antioxidant, anti-glycation, sorbitol accumulation and inhibition of aldose reductase activity.

    Science.gov (United States)

    Gupta, Pankaj; Jain, Vivek; Pareek, Ashutosh; Kumari, Preeti; Singh, Randhir; Agarwal, Priyanka; Sharma, Veena

    2017-07-01

    Rising popularity of phytomedicines in various diseased conditions have strengthened the significance of plant-research and evaluation of phytoextracts in clinical manifestations. Pterocarpus marsupium Roxb., a medicinal plant, known for its anti-oxidant and anti-diabetic activity is a rich source of phytochemicals with antihyperglycemic and antihyperlipidemic activities. However, its possible role in diabetic complications is not evaluated yet. The present study explores the possible role of alcoholic extract of heartwood of P. marsupium in the treatment of long-term diabetic complications. The alcoholic extract of P. marsupium was evaluated for advanced glycation-end-products formation, erythrocyte sorbitol accumulation and rat kidney aldose reductase enzyme inhibition at the concentration of 25-400 μg/ml using in-vitro bioassays. Also the phytoextract at the concentration of 10-320 μg/ml was evaluated for its antioxidant potential by in-vitro antioxidant assays which includes, determination of total phenol content; reducing power assay; nitric oxide scavenging activity; superoxide radical scavenging activity; total antioxidant capacity; total flavonoid content; DPPH scavenging activity; and hydrogen peroxide scavenging activity. The alcoholic extract of P. marsupium across varying concentrations showed inhibitory effect as evident by IC50 on advanced glycation-end-products formation (55.39 μg/ml), sorbitol accumulation (151.00 μg/ml) and rat kidney aldose reductase (195.88 μg/ml). The phytoextract also exhibited high phenolic and flavonoid contents with promising antioxidant potential against the antioxidant assays evaluated. The present investigation suggests that the phytoextract showed prominent antioxidant, antiglycation property and, inhibited accumulation of sorbitol and ALR enzyme, thus promising a beneficial role in reducing/delaying diabetic complications.

  14. Automatic extraction of gene ontology annotation and its correlation with clusters in protein networks

    Directory of Open Access Journals (Sweden)

    Mazo Ilya

    2007-07-01

    Full Text Available Abstract Background Uncovering cellular roles of a protein is a task of tremendous importance and complexity that requires dedicated experimental work as well as often sophisticated data mining and processing tools. Protein functions, often referred to as its annotations, are believed to manifest themselves through topology of the networks of inter-proteins interactions. In particular, there is a growing body of evidence that proteins performing the same function are more likely to interact with each other than with proteins with other functions. However, since functional annotation and protein network topology are often studied separately, the direct relationship between them has not been comprehensively demonstrated. In addition to having the general biological significance, such demonstration would further validate the data extraction and processing methods used to compose protein annotation and protein-protein interactions datasets. Results We developed a method for automatic extraction of protein functional annotation from scientific text based on the Natural Language Processing (NLP technology. For the protein annotation extracted from the entire PubMed, we evaluated the precision and recall rates, and compared the performance of the automatic extraction technology to that of manual curation used in public Gene Ontology (GO annotation. In the second part of our presentation, we reported a large-scale investigation into the correspondence between communities in the literature-based protein networks and GO annotation groups of functionally related proteins. We found a comprehensive two-way match: proteins within biological annotation groups form significantly denser linked network clusters than expected by chance and, conversely, densely linked network communities exhibit a pronounced non-random overlap with GO groups. We also expanded the publicly available GO biological process annotation using the relations extracted by our NLP technology

  15. Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2

    Directory of Open Access Journals (Sweden)

    Hoffman Seth A

    2012-02-01

    Full Text Available Abstract Background New interventions are required to optimally and sustainably control the Anopheles sp. mosquitoes that transmit malaria and filariasis. The mosquito olfactory system is important in host seeking (transmission and mate finding (reproduction. Understanding olfactory function could lead to development of control strategies based on repelling parasite-carrying mosquitoes or attracting them into a fatal trap. Findings Our initial focus is on odorant binding proteins with differential transcript accumulation between female and male mosquitoes. We report that the odorant binding protein, OBP2 (AGAP003306, had increased expression in the antennae of female vs. male Anopheles gambiae sensu stricto (G3 strain. The increased expression in antennae of females of this gene by quantitative RT-PCR was 4.2 to 32.3 fold in three independent biological replicates and two technical replicate experiments using A. gambiae from two different laboratories. OBP2 is a member of the vast OBP superfamily of insect odorant binding proteins and belongs to the predominantly dipteran clade that includes the Culex oviposition kairomone-binding OBP1. Phylogenetic analysis indicates that its orthologs are present across culicid mosquitoes and are likely to play a conserved role in recognizing a molecule that might be critical for female behavior. Conclusions OBP2 has increased mRNA transcript accumulation in the antennae of female as compared to male A. gambiae. This molecule and related molecules may play an important role in female mosquito feeding and breeding behavior. This finding may be a step toward providing a foundation for understanding mosquito olfactory requirements and developing control strategies based on reducing mosquito feeding and breeding success.

  16. Extracting protein alignment models from the sequence database.

    Science.gov (United States)

    Neuwald, A F; Liu, J S; Lipman, D J; Lawrence, C E

    1997-05-01

    Biologists often gain structural and functional insights into a protein sequence by constructing a multiple alignment model of the family. Here a program called Probe fully automates this process of model construction starting from a single sequence. Central to this program is a powerful new method to locate and align only those, often subtly, conserved patterns essential to the family as a whole. When applied to randomly chosen proteins, Probe found on average about four times as many relationships as a pairwise search and yielded many new discoveries. These include: an obscure subfamily of globins in the roundworm Caenorhabditis elegans ; two new superfamilies of metallohydrolases; a lipoyl/biotin swinging arm domain in bacterial membrane fusion proteins; and a DH domain in the yeast Bud3 and Fus2 proteins. By identifying distant relationships and merging families into superfamilies in this way, this analysis further confirms the notion that proteins evolved from relatively few ancient sequences. Moreover, this method automatically generates models of these ancient conserved regions for rapid and sensitive screening of sequences.

  17. Extraction of proteins with ionic liquid aqueous two-phase system based on guanidine ionic liquid.

    Science.gov (United States)

    Zeng, Qun; Wang, Yuzhi; Li, Na; Huang, Xiu; Ding, Xueqin; Lin, Xiao; Huang, Songyun; Liu, Xiaojie

    2013-11-15

    Eight kinds of green ionic liquids were synthesized, and an ionic liquid aqueous two-phase system (ILATPS) based on 1,1,3,3-tetramethylguandine acrylate (TMGA) guanidine ionic liquid was first time studied for the extraction of proteins. Single factor experiments proved that the extraction efficiency of bovine serum albumin (BSA) was influenced by the mass of IL, K2HPO4 and BSA, also related to the separation time and temperature. The optimum conditions were determined through orthogonal experiment by the five factors described above. The results showed that under the optimum conditions, the extraction efficiency could reach up to 99.6243%. The relative standard deviations (RSD) of extraction efficiencies in precision experiment, repeatability experiment and stability experiment were 0.8156% (n=5), 1.6173% (n=5) and 1.6292% (n=5), respectively. UV-vis and FT-IR spectra confirmed that there were no chemical interactions between BSA and ionic liquid in the extraction process, and the conformation of the protein was not changed after extraction. The conductivity, DLS and TEM were combined to investigate the microstructure of the top phase and the possible mechanism for the extraction. The results showed that hydrophobic interaction, hydrogen bonding interaction and the salt out effect played important roles in the transferring process, and the aggregation and embrace phenomenon was the main driving force for the separation. All these results proved that guanidine ionic liquid-based ATPSs have the potential to offer new possibility in the extraction of proteins.

  18. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    Science.gov (United States)

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Protein precipitating capacity and antioxidant activity of Turkish Tombul hazelnut phenolic extract and its fractions.

    Science.gov (United States)

    Pelvan Pelitli, Ebru; Janiak, Michał Adam; Amarowicz, Ryszard; Alasalvar, Cesarettin

    2017-03-01

    Natural (raw) hazelnut was extracted with 80% (v/v) acetone to obtain crude phenolic extract that was then fractionated for elution of low-molecular weight (LMW) and high-molecular weight (HMW) fractions. LMW fraction was further purified (LWM-FP) to remove sugars and organic acids. The crude extract and its fractions were determined by measuring their protein precipitating capacity (PPC) using two different proteins [bovine serum albumin (BSA) and gelatin], molecular weights, total phenolics, condensed tannins, and various antioxidant activities. Significant differences (p<0.05) existed in the contents of total phenolics, condensed tannins, antioxidant activities, and PPC among the crude extract and fractions, albeit to different extends. BSA and gelatin was effectively precipitated by HMW fraction. HMW fraction had the highest total phenolics, condensed tannins, and antioxidant activities, followed by crude extract, LWM-FP, and LMW, respectively. The present study suggests that HMW fraction could be utilised as a source of polyphenols for the food industry.

  20. Extraction of detergents from hydrophobic proteins with isopentanol: application to electrophoretic analysis of photosynthetic bacterial hydrophobic proteins.

    Science.gov (United States)

    Salcedo, J R; Hernández, R S; Célis, H

    1983-07-15

    The method for extracting Triton X-100 used by I. H. Mather and C. B. Tampling [Anal. Biochem. 93, 139-142 (1979)], has been extended to other detergents of different charge and chemical nature. All the detergents tested can be extracted with isopentanol in conditions in which not more than 8% of hydrophobic or hydrophilic protein is lost from the water phase. The removal of detergent from reaction centers and light harvesting protein-pigment complexes of photosynthetic bacteria, eliminates the artifacts of oligomers when analyzed by sodium dodecyl sulfate-gel electrophoresis.

  1. Characteristics of extraction and functionality of protein from tomato pomace produced with different industrial processing methods

    Science.gov (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...

  2. An Approach to Protein Name Extraction Using Heuristics and a Dictionary.

    Science.gov (United States)

    Seki, Kazuhiro; Mostafa, Javed

    2003-01-01

    Proposes a method for protein name extraction from biological texts. The method exploits hand-crafted rules based on heuristics and a set of protein names (dictionary). The approach avoids use of natural language processing tools so as to improve processing speed. Evaluation experiments were conducted in terms of: accuracy, generalizability, and…

  3. Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal

    Science.gov (United States)

    Enzymatic hydrolysis was performed for extracting protein to prepare umami taste amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing. Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of DTSM, protein concentration and fr...

  4. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

    Science.gov (United States)

    Adams, G. R.; Haddad, F.

    1996-01-01

    Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

  5. The respective effects of soil heavy metal fractions by sequential extraction procedure and soil properties on the accumulation of heavy metals in rice grains and brassicas.

    Science.gov (United States)

    Xiao, Ling; Guan, Dongsheng; Peart, M R; Chen, Yujuan; Li, Qiqi

    2017-01-01

    This study was carried out to examine heavy metal accumulation in rice grains and brassicas and to identify the different controls, such as soil properties and soil heavy metal fractions obtained by the Community Bureau of Reference (BCR) sequential extraction, in their accumulation. In Guangdong Province, South China, rice grain and brassica samples, along with their rhizospheric soil, were collected from fields on the basis of distance downstream from electroplating factories, whose wastewater was used for irrigation. The results showed that long-term irrigation using the electroplating effluent has not only enriched the rhizospheric soil with Cd, Cr, Cu, and Zn but has also increased their mobility and bioavailability. The average concentrations of Cd and Cr in rice grains and brassicas from closest to the electroplating factories were significantly higher than those from the control areas. Results from hybrid redundancy analysis (hRDA) and redundancy analysis (RDA) showed that the BCR fractions of soil heavy metals could explain 29.0 and 46.5 % of total eigenvalue for heavy metal concentrations in rice grains and brassicas, respectively, while soil properties could only explain 11.1 and 33.4 %, respectively. This indicated that heavy metal fractions exerted more control upon their concentrations in rice grains and brassicas than soil properties. In terms of metal interaction, an increase of residual Zn in paddy soil or a decrease of acid soluble Cd in the brassica soil could enhance the accumulation of Cd, Cu, Cr, and Pb in both rice grains and brassicas, respectively, while the reducible or oxidizable Cd in soil could enhance the plants' accumulation of Cr and Pb. The RDA showed an inhibition effect of sand content and CFO on the accumulation of heavy metals in rice grains and brassicas. Moreover, multiple stepwise linear regression could offer prediction for Cd, Cu, Cr, and Zn concentrations in the two crops by soil heavy metal fractions and soil properties.

  6. Calcium Sulfate with Stearic Acid as an Encouraging Carrier for Reindeer Bone Protein Extract

    Directory of Open Access Journals (Sweden)

    Pekka Jalovaara

    2011-07-01

    Full Text Available Various bone proteins and growth factors in specific concentrations are required for bone formation. If the body cannot produce sufficient quantities of these factors, bone trauma can be healed with an implant that includes the required factors in a carrier. This study was designed to evaluate various calcium salt candidates that can be used as carrier with reindeer bone protein extract to induce ectopic bone formation in the muscle pouch model of mouse. The bone protein extract was either impregnated into the disc form of carrier or mixed with carrier powder before implantation. The radiographic analysis indicated increased bone formation in all of the active groups containing the bone protein extract compared to the controls within 21 days follow-up. The highest bone formation was seen in the group with calcium sulfate with stearic acid where new bone and calcified cartilage were clearly visible. The greatest bone formation occurred in the groups that had bone protein extract readily available. This indicates that the bone forming factors in sufficient concentrations are required at the early stage of bone formation. The calcium sulfate with stearic acid was the most suitable and effective carrier for reindeer bone protein extract.

  7. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves.

    Science.gov (United States)

    Zhang, Chen; Sanders, Johan P M; Xiao, Ting T; Bruins, Marieke E

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-protein components obtained at various conditions. Alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, and organic acids were extracted before protein extraction, which was followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin were both linear to protein yield. The yields of these two components were 80% and 25% respectively when 95% protein was extracted, which indicated that RGI pectin is more likely to be the key limitation to leaf protein extraction. An integrated biorefinery was designed based on these results.

  8. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves

    Science.gov (United States)

    Zhang, Chen; Sanders, Johan P. M.; Xiao, Ting T.; Bruins, Marieke E.

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-protein components obtained at various conditions. Alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, and organic acids were extracted before protein extraction, which was followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin were both linear to protein yield. The yields of these two components were 80% and 25% respectively when 95% protein was extracted, which indicated that RGI pectin is more likely to be the key limitation to leaf protein extraction. An integrated biorefinery was designed based on these results. PMID:26200774

  9. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves.

    Directory of Open Access Journals (Sweden)

    Chen Zhang

    Full Text Available Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-protein components obtained at various conditions. Alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, and organic acids were extracted before protein extraction, which was followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin were both linear to protein yield. The yields of these two components were 80% and 25% respectively when 95% protein was extracted, which indicated that RGI pectin is more likely to be the key limitation to leaf protein extraction. An integrated biorefinery was designed based on these results.

  10. Effects of Free Anthraquinones Extract from the Rhubarb on Cell Proliferation and Accumulation of Extracellular Matrix in High Glucose Cultured-Mesangial Cells

    Science.gov (United States)

    Wang, Jianyun; Fang, Hui; Dong, Bingzheng; Wang, Dongdong; Li, Yan; Chen, Xiao; Chen, Lijuan; Wei, Tong

    2015-01-01

    Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of free anthraquinones (FARs) extract, which was extracted from the rhubarb and purified by macroporous resin DM130 with gradient mixtures of ethanol/water as the lelution solvents, in high glucose-cultured glomerular mesangial cells (MCs). The cell proliferation was determined by CCK-8 assay, the levels of TGF-β1, CTGF, ColIV and FN proteins in the supernatant of MCs were measured by ELISA assays, and the mRNA levels of these four genes were detected by RT-PCR. The results showed that the increased proliferation of MCs, the mRNA levels and protein expression of TGF-β1, CTGF, ColIV and FN induced by high glucose were inhibited after the treatment with the FARs extract. This indicated that FARs extract could inhibit cell proliferation and the expression of main extracellular matrix induced by high glucose in MCs. The FARs extract exhibited potential values for prophylaxis and therapy of DN. PMID:26557014

  11. Alkali-aided protein extraction from chicken dark meat: textural properties and color characteristics of recovered proteins.

    Science.gov (United States)

    Omana, D A; Moayedi, V; Xu, Y; Betti, M

    2010-05-01

    Textural properties, water-holding capacity, and color characteristics of alkali-extracted chicken dark meat have been studied. Alkali extraction was carried out at 4 different pH values (10.5, 11.0, 11.5, and 12.0). At higher pH of extraction, cooking loss and water loss were found to be significantly decreased (P extraction pH values. Protein samples extracted at higher pH values were found to be harder, and the maximum (4,956 g of force) value was shown by samples prepared at pH 11.5. Chewiness values were significantly increased (P protein samples extracted at pH values of 11.5 and 12.0. Dynamic viscoelastic behavior of samples was assessed in the temperature range of 7 to 100 degrees C. The dynamic viscoelastic behavior of raw chicken dark meat as revealed by storage modulus indicated considerable gel-forming ability. The maximum storage modulus (G') value of 439 kPa was measured at 66.7 degrees C. Storage modulus was found to decrease for the recovered protein samples and be lowest at higher pH values. However, the recovered protein samples did show substantial gel-forming ability when stored with cryoprotectants. Tan delta values denoted 2 clear transitions for raw dark meat; however, only 1 major transition at 50.1 degrees C was evident for pH-treated samples, probably reflecting the loss of collagen in processing. In conclusion, this process of protein recovery may offer the possibility to use the underused poultry resources for preparation of functional foods.

  12. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

    Directory of Open Access Journals (Sweden)

    Yuh-Ching Twu

    2016-07-01

    Full Text Available In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2 protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1-induced collagen type 1 α1 (Col1a1, α-smooth muscle actin (α-SMA expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

  13. Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation.

    Science.gov (United States)

    Twu, Yuh-Ching; Lee, Tzong-Shyuan; Lin, Yun-Lian; Hsu, Shih-Ming; Wang, Yuan-Hsi; Liao, Chia-Yu; Wang, Chung-Kwe; Liang, Yu-Chih; Liao, Yi-Jen

    2016-07-13

    In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

  14. SGT1 interacts with the Prf resistance protein and is required for Prf accumulation and Prf-mediated defense signaling.

    Science.gov (United States)

    Kud, Joanna; Zhao, Zhulu; Du, Xinran; Liu, Yule; Zhao, Yun; Xiao, Fangming

    2013-02-15

    The highly conserved eukaryotic co-chaperone SGT1 (suppressor of the G2 allele of skp1) is an important signaling component of plant defense responses and positively regulates disease resistance conferred by many resistance (R) proteins. In this study, we investigated the contribution of SGT1 in the Prf-mediated defense responses in both Nicotiana benthamiana and tomato (Solanum lycopersicum). SGT1 was demonstrated to interact with Prf in plant cells by co-immunoprecipitation. The requirement of SGT1 in the accumulation of Prf or autoactive Prf(D1416V) was determined by the degradation of these proteins in N. benthamiana, in which SGT1 was repressed by virus-induced gene silencing (VIGS). Pseudomonas pathogen assay on the SGT1-silenced tomato plants implicates SGT1 is required for the Prf-mediated full resistance to Pseudomonas syringae pv. tomato (Pst). These results suggest that, in both N. benthamiana and tomato, SGT1 contributes to the Prf-mediated defense responses by stabilizing Prf protein via its co-chaperone activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G Protein, Gαs.

    Science.gov (United States)

    Erb, Samuel J; Schappi, Jeffrey M; Rasenick, Mark M

    2016-09-16

    Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify Gαs localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of Gαs to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics.

  16. Accumulation of pathological prion protein PrPSc in the skin of animals with experimental and natural scrapie.

    Directory of Open Access Journals (Sweden)

    Achim Thomzig

    2007-05-01

    Full Text Available Prion infectivity and its molecular marker, the pathological prion protein PrP(Sc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrP(Sc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles. Here, we address the question of whether prions target the skin and show widespread PrP(Sc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrP(Sc was detected before the onset of symptoms, but the bulk of skin-associated PrP(Sc accumulated in the clinical phase. PrP(Sc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrP(Sc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrP(Sc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.

  17. Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

    Science.gov (United States)

    Vian, A.; Henry-Vian, C.; Davies, E.

    1999-01-01

    It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

  18. Host-derived smooth muscle cells accumulate in cardiac allografts: role of inflammation and monocyte chemoattractant protein 1.

    Directory of Open Access Journals (Sweden)

    Piotr Religa

    Full Text Available Transplant arteriosclerosis is characterized by inflammation and intimal thickening caused by accumulation of smooth muscle cells (SMCs both from donor and recipient. We assessed the relationship between clinical factors and the presence of host-derived SMCs in 124 myocardial biopsies from 26 consecutive patients who received hearts from opposite-sex donors. Clinical and demographic information was obtained from the patients' medical records. Host-derived SMCs accounted for 3.35+/-2.3% of cells in arterioles (range, 0.08-12.51%. As shown by linear regression analysis, an increased number of SMCs was associated with rejection grade (mean, 1.41+/-1.03, p = 0.034 and the number of leukocytes (19.1+/-12.7 per 20 high-power fields, p = 0.01. The accumulation of host-derived SMCs was associated with an increased number of leukocytes in the allografts. In vitro, monocyte chemoattractant protein 1 (MCP-1 released from leukocytes was crucial for SMC migration. After heart allotransplantation, mice treated with MCP-1-specific antibodies had significantly fewer host-derived SMCs in the grafts than mice treated with isotypic antibody controls. We conclude that the number of host-derived SMCs in human cardiac allografts is associated with the rejection grade and that MCP-1 may play pivotal role in recruiting host-derived SMCs into cardiac allografts.

  19. Host-Derived Smooth Muscle Cells Accumulate in Cardiac Allografts: Role of Inflammation and Monocyte Chemoattractant Protein 1

    Science.gov (United States)

    Bojakowski, Krzysztof; Soin, Joanna; Nozynski, Jerzy; Zakliczynski, Michal; Gaciong, Zbigniew; Zembala, Marian; Söderberg-Nauclér, Cecilia

    2009-01-01

    Transplant arteriosclerosis is characterized by inflammation and intimal thickening caused by accumulation of smooth muscle cells (SMCs) both from donor and recipient. We assessed the relationship between clinical factors and the presence of host-derived SMCs in 124 myocardial biopsies from 26 consecutive patients who received hearts from opposite-sex donors. Clinical and demographic information was obtained from the patients' medical records. Host-derived SMCs accounted for 3.35±2.3% of cells in arterioles (range, 0.08–12.51%). As shown by linear regression analysis, an increased number of SMCs was associated with rejection grade (mean, 1.41±1.03, p = 0.034) and the number of leukocytes (19.1±12.7 per 20 high-power fields, p = 0.01). The accumulation of host-derived SMCs was associated with an increased number of leukocytes in the allografts. In vitro, monocyte chemoattractant protein 1 (MCP-1) released from leukocytes was crucial for SMC migration. After heart allotransplantion, mice treated with MCP-1-specific antibodies had significantly fewer host-derived SMCs in the grafts than mice treated with isotypic antibody controls. We conclude that the number of host-derived SMCs in human cardiac allografts is associated with the rejection grade and that MCP-1 may play pivotal role in recruiting host-derived SMCs into cardiac allografts. PMID:19142231

  20. Use of sequential chemical extractions to purify nuclear membrane proteins for proteomics identification.

    Science.gov (United States)

    Korfali, Nadia; Fairley, Elizabeth A L; Swanson, Selene K; Florens, Laurence; Schirmer, Eric C

    2009-01-01

    The nuclear envelope (NE) is a double membrane system that is both a part of the endoplasmic reticulum and part of the nucleus. As its constituent proteins tend to be highly complexed with nuclear and cytoplasmic components, it is notoriously difficult to purify. Two methods can reduce this difficulty for the identification of nuclear membrane proteins: comparison to contaminating membranes and chemical extractions to enrich for certain groups of proteins. The purification of nuclear envelopes and contaminating microsomal membranes is described here along with procedures for chemical extraction using salt and detergent, chaotropes, or alkaline solutions. Each extraction method enriches for different combinations of nuclear envelope proteins. Finally, we describe the analysis of these fractions with MudPIT, a proteomics methodology that avoids gel extraction of bands to facilitate identification of minor proteins and membrane proteins that do not resolve well on gels. Together these three approaches can significantly increase the output of proteomics studies aimed at identifying the protein complement of subcellular membrane systems.

  1. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain.

    Science.gov (United States)

    Arnold, J E; Tipler, C; Laszlo, L; Hope, J; Landon, M; Mayer, R J

    1995-08-01

    The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

  2. PPI-IRO: A two-stage method for protein-protein interaction extraction based on interaction relation ontology

    KAUST Repository

    Li, Chuanxi

    2014-01-01

    Mining Protein-Protein Interactions (PPIs) from the fast-growing biomedical literature resources has been proven as an effective approach for the identifi cation of biological regulatory networks. This paper presents a novel method based on the idea of Interaction Relation Ontology (IRO), which specifi es and organises words of various proteins interaction relationships. Our method is a two-stage PPI extraction method. At fi rst, IRO is applied in a binary classifi er to determine whether sentences contain a relation or not. Then, IRO is taken to guide PPI extraction by building sentence dependency parse tree. Comprehensive and quantitative evaluations and detailed analyses are used to demonstrate the signifi cant performance of IRO on relation sentences classifi cation and PPI extraction. Our PPI extraction method yielded a recall of around 80% and 90% and an F1 of around 54% and 66% on corpora of AIMed and Bioinfer, respectively, which are superior to most existing extraction methods. Copyright © 2014 Inderscience Enterprises Ltd.

  3. 14C Analysis of protein extracts from Bacillus spores.

    Science.gov (United States)

    Cappuccio, Jenny A; Falso, Miranda J Sarachine; Kashgarian, Michaele; Buchholz, Bruce A

    2014-07-01

    Investigators of bioagent incidents or interdicted materials need validated, independent analytical methods that will allow them to distinguish between recently made bioagent samples versus material drawn from the archives of a historical program. Heterotrophic bacteria convert the carbon in their food sources, growth substrate or culture media, into the biomolecules they need. The F(14)C (fraction modern radiocarbon) of a variety of media, Bacillus spores, and separated proteins from Bacillus spores was measured by accelerator mass spectrometry (AMS). AMS precisely measures F(14)C values of biological materials and has been used to date the synthesis of biomaterials over the bomb pulse era (1955 to present). The F(14)C of Bacillus spores reflects the radiocarbon content of the media in which they were grown. In a survey of commercial media we found that the F(14)C value indicated that carbon sources for the media were alive within about a year of the date of manufacture and generally of terrestrial origin. Hence, bacteria and their products can be dated using their (14)C signature. Bacillus spore samples were generated onsite with defined media and carbon free purification and also obtained from archived material. Using mechanical lysis and a variety of washes with carbon free acids and bases, contaminant carbon was removed from soluble proteins to enable accurate (14)C bomb-pulse dating. Since media is contemporary, (14)C bomb-pulse dating of isolated soluble proteins can be used to distinguish between historical archives of bioagents and those produced from recent media.

  4. Magnetic solid-phase extraction of protein with deep eutectic solvent immobilized magnetic graphene oxide nanoparticles.

    Science.gov (United States)

    Xu, Kaijia; Wang, Yuzhi; Ding, Xueqin; Huang, Yanhua; Li, Na; Wen, Qian

    2016-01-01

    As a new type of green solvent, four kinds of choline chloride (ChCl)-based deep eutectic solvents (DESs) have been synthesized, and then a core-shell structure magnetic graphene oxide (Fe3O4-NH2@GO) nanoparticles have been prepared and coated with the ChCl-based DESs. Magnetic solid-phase extraction (MSPE) based Fe3O4-NH2@GO@DES was studied for the first time for the extraction of proteins. The characteristic results of vibrating sample magnetometer (VSM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA) and field emission scanning electron microscopy (FESEM) indicated the successful preparation of Fe3O4-NH2@GO@DES. The concentrations of proteins in studies were determined by a UV-vis spectrophotometer. The advantages of Fe3O4-NH2@GO@DES in protein extraction were compared with Fe3O4-NH2@GO and Fe3O4-NH2, and Fe3O4-NH2@GO@ChCl-glycerol was selected as the suitable extraction solvent. The influence factors of the extraction process such as the pH value, the temperature, the extraction time, the concentration of protein and the amount of Fe3O4-NH2@GO@ChCl-glycerol were evaluated. Desorption experimental result showed 98.73% of BSA could be eluted from the solid extractant with 0.1 mol/L Na2HPO4 solution contained 1 mol/L NaCl. Besides, the conformation of BSA was not changed during the elution by the investigation of circular dichromism (CD) spectra. Furthermore, the analysis of real sample demonstrated that the prepared magnetic nanoparticles did have extraction ability on proteins in bovine whole blood.

  5. Accumulation of Glycoconjugates of 3-Methyl-4-hydroxyoctanoic Acid in Fruits, Leaves, and Shoots of Vitis vinifera cv. Monastrell following Foliar Applications of Oak Extract or Oak Lactone.

    Science.gov (United States)

    Pardo-Garcia, Ana I; Wilkinson, Kerry L; Culbert, Julie A; Lloyd, Natoiya D R; Alonso, Gonzalo L; Salinas, M Rosario

    2015-05-13

    Grapevines are capable of absorbing volatile compounds present in the vineyard during the growing season, and in some cases, volatiles have been found to accumulate in fruits or leaves in glycoconjugate forms, that is, with one or more sugar moieties attached. The presence of oak lactone in wine is usually attributable to oak maturation, but oak lactone has been detected in wines made with fruit from grapevines treated with oak extract or oak lactone. This study investigated the accumulation of glycoconjugates of 3-methyl-4-hydroxyoctanoic acid (i.e., the ring-opened form of oak lactone) in the fruits, leaves, and shoots of Monastrell grapevines following foliar application of either oak extract or oak lactone at approximately 7 days postveraison. Fruits, leaves, and shoots were collected at three different time points, including at maturity. The oak lactone content of fruit was determined by gas chromatography-mass spectrometry, with declining concentrations observed in fruit from grapevines treated with oak lactone with ripening. The concentrations of a β-d-glucopyranoside of 3-methyl-4-hydroxyoctanoic acid in fruits, leaves, and shoots was determined by liquid chromatography-tandem mass spectrometry, with the highest oak lactone glucoside levels observed in leaves of grapevines treated with oak lactone. A glucose-glucose disaccharide was also tentatively identified. These results demonstrate both ring-opening and glycosylation of oak lactone occurred after experimental treatments were imposed.

  6. Comparison between BCR sequential extraction and geo-accumulation method to evaluate metal mobility in sediments of Dongting Lake, Central China

    Science.gov (United States)

    Yao, Zhigang

    2008-02-01

    The form in which a metal exists strongly influences its mobility and thus, the effects on the environment. Operational methods of speciation analysis, such as the use of sequential extraction procedures, are commonly applied. The Dongting Lake, the second largest fresh-water lake in China, contains three China wetlands of international importance, the East Dongting Lake, South Dongting Lake, and West Dongting Lake. In this work, an optimized BCR sequential extraction procedure was used to assess the environmental risk of Cd, Cr, Cu, Ni, Pb and Zn in contaminated sediment of the Dongting Lake. The procedure was evaluated by using a certified reference material, BCR701. The results of the partitioning study indicated that in the lake sediments, more easily mobilized forms (acid exchangeable) were predominant for Cd, particularly in the samples from the East Dongting Lake. In contrast, the largest amount of Pb was associated with the iron and manganese oxide fractions and Cu, Zn, Cr, and Ni analyzed were mainly distributed in residual phase at an average percentage greater than 60% of the total metals. The potential risk to the lake’s water contamination was highest in the East Dongting Lake based on the calculated contamination factors. On the other hand, the total metal content was determined as well by inductively coupled plasma and mass spectrometry (ICP-MS) and assessed by using geo-accumulation index. The assessment results using geo-accumulation index were compared with the information on metal speciation. Both were correspondent with each other.

  7. Mechanism of the Inhibitory Effects of Eucommia ulmoides Oliv. Cortex Extracts (EUCE in the CCl4-Induced Acute Liver Lipid Accumulation in Rats

    Directory of Open Access Journals (Sweden)

    Chang-Feng Jin

    2013-01-01

    Full Text Available Eucommia ulmoides Oliv. (EU has been used for treatment of liver diseases. The protective effects of Eucommia Ulmoides Oliv. cortex extracts (EUCE on the carbon tetrachloride- (CCl4- induced hepatic lipid accumulation were examined in this study. Rats were orally treated with EUCE in different doses prior to an intraperitoneal injection of 1 mg/kg CCl4. Acute injection of CCl4 decreased plasma triglyceride but increased hepatic triglyceride and cholesterol as compared to control rats. On the other hand, the pretreatment with EUCE diminished these effects at a dose-dependent manner. CCl4 treatment decreased glutathione (GSH and increased malondialdehyde (MDA accompanied by activated P450 2E1. The pretreatment with EUCE significantly improved these deleterious effects of CCl4. CCl4 treatment increased P450 2E1 activation and ApoB accumulation. Pretreatment with EUCE reversed these effects. ER stress response was significantly increased by CCl4, which was inhibited by EUCE. One of the possible ER stress regulatory mechanisms, lysosomal activity, was examined. CCl4 reduced lysosomal enzymes that were reversed with the EUCE. The results indicate that oral pretreatment with EUCE may protect liver against CCl4-induced hepatic lipid accumulation. ER stress and its related ROS regulation are suggested as a possible mechanism in the antidyslipidemic effect of EUCE.

  8. Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients.

    Science.gov (United States)

    Viteri, Gabriela; Chung, Youn Wook; Stadtman, Earl R

    2010-01-01

    The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging.

  9. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

    Science.gov (United States)

    Adams, G. R.; Haddad, F.

    1996-01-01

    Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P Muscle IGF-1 peptide levels peaked at 3 (normal) and 7 (HX) days of overloading with maximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases in muscle IGF-1 preceded the hypertrophic response. Total DNA content of the overloaded Plant increased in both groups. There was a strong positive relationship between IGF-1 peptide and DNA content in the overloaded Plant from both groups. These results indicate that 1) the muscles from rats with both normal and severely depressed systemic levels of IGF-1 respond to functional overload with an increase in local IGF-1 expression and 2) this elevated IGF-1 may be contributing to the hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

  10. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

    Science.gov (United States)

    Adams, G. R.; Haddad, F.

    1996-01-01

    Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P peptide levels peaked at 3 (normal) and 7 (HX) days of overloading with maximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases in muscle IGF-1 preceded the hypertrophic response. Total DNA content of the overloaded Plant increased in both groups. There was a strong positive relationship between IGF-1 peptide and DNA content in the overloaded Plant from both groups. These results indicate that 1) the muscles from rats with both normal and severely depressed systemic levels of IGF-1 respond to functional overload with an increase in local IGF-1 expression and 2) this elevated IGF-1 may be contributing to the hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

  11. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

    Science.gov (United States)

    Adams, G. R.; Haddad, F.

    1996-01-01

    Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P Muscle IGF-1 peptide levels peaked at 3 (normal) and 7 (HX) days of overloading with maximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases in muscle IGF-1 preceded the hypertrophic response. Total DNA content of the overloaded Plant increased in both groups. There was a strong positive relationship between IGF-1 peptide and DNA content in the overloaded Plant from both groups. These results indicate that 1) the muscles from rats with both normal and severely depressed systemic levels of IGF-1 respond to functional overload with an increase in local IGF-1 expression and 2) this elevated IGF-1 may be contributing to the hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

  12. A realistic assessment of methods for extracting gene/protein interactions from free text

    Directory of Open Access Journals (Sweden)

    Shepherd Adrian J

    2009-07-01

    Full Text Available Abstract Background The automated extraction of gene and/or protein interactions from the literature is one of the most important targets of biomedical text mining research. In this paper we present a realistic evaluation of gene/protein interaction mining relevant to potential non-specialist users. Hence we have specifically avoided methods that are complex to install or require reimplementation, and we coupled our chosen extraction methods with a state-of-the-art biomedical named entity tagger. Results Our results show: that performance across different evaluation corpora is extremely variable; that the use of tagged (as opposed to gold standard gene and protein names has a significant impact on performance, with a drop in F-score of over 20 percentage points being commonplace; and that a simple keyword-based benchmark algorithm when coupled with a named entity tagger outperforms two of the tools most widely used to extract gene/protein interactions. Conclusion In terms of availability, ease of use and performance, the potential non-specialist user community interested in automatically extracting gene and/or protein interactions from free text is poorly served by current tools and systems. The public release of extraction tools that are easy to install and use, and that achieve state-of-art levels of performance should be treated as a high priority by the biomedical text mining community.

  13. Characterization of green-tissue protein extract from alfalfa (Medicago sativa) exploiting a 3-D technique.

    Science.gov (United States)

    Aguilar, Oscar; Glatz, Charles E; Rito-Palomares, Marco

    2009-09-01

    There is a growing interest of pharmaceutical companies for plant-based production systems. To facilitate the general acceptance of plants as bioreactors, the establishment of efficient downstream operations is critical. It has been proposed that a better understanding of the properties of the contaminant proteins can benefit downstream processing design and operation. The coupled application of 2-DE with aqueous two-phase partitioning has been suggested as a practical 3-D method to characterize potential contaminant proteins from plant extracts. The application of this novel 3-D approach to a complex protein extract from alfalfa (Medicago sativa) containing a model recombinant protein (human granulocyte colony stimulating factor (hG-CSF)) resulted in the quantification of 55 protein spots. The 3-D properties (M(r), pI, and K(p)) obtained for 17 proteins comprising 69% of the alfalfa proteins, allowed the proposal of a prefractionation step as well as the identification of the target molecule (rG-CSF) from bulk of alfalfa proteins. The information obtained from this experimental approach was useful for the identification of the potential contaminant proteins that will occur in alfalfa when this plant is used as a host for recombinant proteins. Additionally, this method will assist in the design of adequate purification strategies for recombinant proteins expressed in alfalfa green tissue.

  14. High-throughput preparation methods of crude extract for robust cell-free protein synthesis.

    Science.gov (United States)

    Kwon, Yong-Chan; Jewett, Michael C

    2015-03-02

    Crude extract based cell-free protein synthesis (CFPS) has emerged as a powerful technology platform for high-throughput protein production and genetic part characterization. Unfortunately, robust preparation of highly active extracts generally requires specialized and costly equipment and can be labor and time intensive. Moreover, cell lysis procedures can be hard to standardize, leading to different extract performance across laboratories. These challenges limit new entrants to the field and new applications, such as comprehensive genome engineering programs to improve extract performance. To address these challenges, we developed a generalizable and easily accessible high-throughput crude extract preparation method for CFPS based on sonication. To validate our approach, we investigated two Escherichia coli strains: BL21 Star™ (DE3) and a K12 MG1655 variant, achieving similar productivity (defined as CFPS yield in g/L) by varying only a few parameters. In addition, we observed identical productivity of cell extracts generated from culture volumes spanning three orders of magnitude (10 mL culture tubes to 10 L fermentation). We anticipate that our rapid and robust extract preparation method will speed-up screening of genomically engineered strains for CFPS applications, make possible highly active extracts from non-model organisms, and promote a more general use of CFPS in synthetic biology and biotechnology.

  15. Method Development for Extraction of Butyrylcholin- esterase using Protein-G Agarose Spin Columns

    Directory of Open Access Journals (Sweden)

    Amruta S. Indapurkar

    2015-01-01

    Full Text Available Butyrylcholinesterase (BuChE is a biomarker of organophosphate (OP poisoning and can be used as a diagnostic marker to measure exposure to OP compounds. The purpose of this study was to develop a method to extract BuChE from human plasma. BuChE was extracted from plasma using the NAb protein-G Agarose Spin Kit. Factors affecting extraction like incubation time, plasma volume and cross-linking of antibodies to agarose beads were evaluated. All samples were analyzed for BuChE activity using the Ellman’s assay. The incubation times of plasma and anti-BuChE antibodies marginally affected the extraction efficiency of BuChE whereas a decrease in plasma volume increased the extraction efficiency. Cross-linking of anti-BuChE antibodies on agarose increased the extraction efficiency. The NAb protein-G Spin Kit can be used successfully to extract BuChE from human plasma. This extraction technique may be coupled to downstream analytical analyses for diagnosing exposure to OP compounds.

  16. Selective separation and enrichment of proteins in aqueous two-phase extraction system

    Institute of Scientific and Technical Information of China (English)

    Feng Qu; Hao Qin; Min Dong; Dong Xu Zhao; Xin Ying Zhao; Jing Hua Zhang

    2009-01-01

    A simple aqueous two-phase extraction system(ATPS)of PEG/phosphate was proposed for selective separation and enrichment of proteins.The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases.Five proteins (bovine serum albumin,Cytochrome C,lysozyme,myoglobin,and trypsin)were used as model proteins to study the effect of phosphate concentration and pH on proteins partition.The PEG/phosphate system was firstly applied to real human saliva and plasma samples,some proteins showed obviously different partition in two phases.The primary results manifest the selective separation and enrichment of proteins in ATPS provided the potential for high abundance proteins depletion in proteomics.

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

    Directory of Open Access Journals (Sweden)

    Elena R. Schroeter

    2016-10-01

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

  18. 14C Analysis of Protein Extracts from Bacillus Spores

    Science.gov (United States)

    Cappucio, Jenny A.; Sarachine Falso, Miranda J.; Kashgarian, Michaele; Buchholz, Bruce A.

    2014-01-01

    Investigators of bioagent incidents or interdicted materials need validated, independent analytical methods that will allow them to distinguish between recently made bioagent samples versus material drawn from the archives of a historical program. Heterotrophic bacteria convert the carbon in their food sources, growth substrate or culture media, into the biomolecules they need. The F14C (fraction modern radiocarbon) of a variety of media, Bacillus spores, and separated proteins from Bacillus spores was measured by accelerator mass spectrometry (AMS). AMS precisely measures F14C values of biological materials and has been used to date the synthesis of biomaterials over the bomb pulse era (1955 to present). The F14C of Bacillus spores reflects the radiocarbon content of the media in which they were grown. In a survey of commercial media we found that the F14C value indicated that carbon sources for the media were alive within about a year of the date of manufacture and generally of terrestrial origin. Hence, bacteria and their products can be dated using their 14C signature. Bacillus spore samples were generated onsite with defined media and carbon free purification and also obtained from archived material. Using mechanical lysis and a variety of washes with carbon free acids and bases, contaminant carbon was removed from soluble proteins to enable accurate 14C bomb-pulse dating. Since media is contemporary, 14C bomb-pulse dating of isolated soluble proteins can be used to distinguish between historical archives of bioagents and those produced from recent media. PMID:24814329

  19. Total protein extraction and 2-D gel electrophoresis methods for Burkholderia species.

    Science.gov (United States)

    Velapatiño, Billie; Zlosnik, James E A; Hird, Trevor J; Speert, David P

    2013-10-15

    The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

  20. Accumulated SET protein up-regulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, Luciana O.; Garcia, Cristiana B.; Matos-Silva, Flavia A. [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Curti, Carlos [Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil); Leopoldino, Andréia M., E-mail: andreiaml@usp.br [Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP (Brazil)

    2014-02-28

    Highlights: • hnRNPK is a new target of SET. • SET regulates hnRNPK. • SET and hnRNPK accumulation promotes tumorigenesis. • SET accumulation is a potential model to study genes regulated by SET-hnRNPK. - Abstract: SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET–hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.

  1. Total Soluble Protein Extraction for Improved Proteomic Analysis of Transgenic Rice Plant Roots.

    Science.gov (United States)

    Raorane, Manish L; Narciso, Joan O; Kohli, Ajay

    2016-01-01

    With the advent of high-throughput platforms, proteomics has become a powerful tool to search for plant gene products of agronomic relevance. Protein extractions using multistep protocols have been shown to be effective to achieve better proteome profiles than simple, single-step extractions. These protocols are generally efficient for above ground tissues such as leaves. However, each step leads to loss of some amount of proteins. Additionally, compounds such as proteases in the plant tissues lead to protein degradation. While protease inhibitor cocktails are available, these alone do not seem to suffice when roots are included in the plant sample. This is obvious given the lack of high molecular weight (HMW) proteins obtained from samples that include root tissue. For protein/proteome analysis of transgenic plant roots or of seedlings, which include root tissue, such pronounced protein degradation is especially undesirable. A facile protein extraction protocol is presented, which ensures that despite the inclusion of root tissues there is minimal loss in total protein components.

  2. Towards plant protein refinery: Review on protein extraction using alkalo and potential enzymatic assistance

    NARCIS (Netherlands)

    Sari, Y.W.; Mulder, W.J.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein extra

  3. Towards plant protein refinery: Review on protein extraction using alkalo and potential enzymatic assistance

    NARCIS (Netherlands)

    Sari, Y.W.; Mulder, W.J.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein

  4. Conformational transition and mass transfer in extraction of proteins by AOT--alcohol--isooctane reverse micellar systems.

    Science.gov (United States)

    Hong, D P; Lee, S S; Kuboi, R

    2000-06-23

    We examined quantitatively the effect of alcohols on protein and reverse micellar structure. We used circular dichroism (CD) to compare the effects of various alcohols on the protein structure, and percolation phenomena to evaluate the effects of various alcohols on reverse micellar structure. Upon the addition of alcohols to the bulk aqueous phase, proteins were denatured significantly, depending on the alcohol species and concentration, suggesting that use of alcohol directly to the stripping solution is not effective in back-extraction processes of proteins. In the present study, a new method, a small amount of alcohol is added to the surfactant-organic solution to improve the back-extraction behaviors of proteins. Practically, in the back-extraction process, the alcohols suppressing the cluster formation of reverse micelles (high value of beta1), remarkably improved the back-extraction behavior of proteins. In addition, the same alcohol molecules showed a positive effect on the rate and fraction of protein back-extraction. From a result of the CD measurement of the back-extracted proteins, it was known that the alcohols added to reverse micellar solution allowed the proteins to back-extract safely without causing structural changes. These results show that the values of beta(t), defined by the variation of percolation processes, and the back-extraction behaviors of proteins have a good relationship, suggesting that the back-extraction processes were controlled by the micellar-micellar and protein-micellar interactions.

  5. HPLC-MS/MS shotgun proteomic research of deer antlers with multiparallel protein extraction methods.

    Science.gov (United States)

    Gao, Liang; Tao, Dingyin; Shan, Yichu; Liang, Zhen; Zhang, Lihua; Huo, Yushu; Zhang, Yukui

    2010-12-15

    Deer antlers mature rapidly in 60 days, and subsequently shed in 5 days with rapid ossification. During this procedure, the function of deer antlers changes significantly. Therefore, the profiling of antler proteome is helpful to discover important growing and shedding regulation proteins, which might be of great significance for studying development and regeneration. In this study, a parallel protein extraction strategy was developed to extract proteins from antlers of red deer with five different lysis solutions, followed by shotgun proteomic analysis by microflow reversed-phase liquid chromatography/electrospray ionization/tandem mass spectrometry (μRPLC-ESI-MS/MS) with a 30 cm-long serially coupled microcolumn. Our experimental results showed that the identified proteins extracted by five kinds of lysis solution were complementary to each other. In total, 416 unique proteins were identified, with relative molecular masses from 2000 to 600,000, and isoelectric points from 3.84 to 11.57. All these results demonstrate that the combination of parallel protein extraction strategy and μRPLC-ESI-MS/MS analysis with serially coupled long microcolumns might be of great significance for comprehensive proteomic research of deer antler.

  6. Comparative Proteomic Analysis of Cotton Fiber Development and Protein Extraction Method Comparison in Late Stage Fibers

    Directory of Open Access Journals (Sweden)

    Hana Mujahid

    2016-02-01

    Full Text Available The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.

  7. A Protein Extract from Chicken Reduces Plasma Homocysteine in Rats

    Directory of Open Access Journals (Sweden)

    Vegard Lysne

    2015-06-01

    Full Text Available The present study aimed to evaluate effects of a water-soluble protein fraction of chicken (CP, with a low methionine/glycine ratio, on plasma homocysteine and metabolites related to homocysteine metabolism. Male Wistar rats were fed either a control diet with 20% w/w casein as the protein source, or an experimental diet where 6, 14 or 20% w/w of the casein was replaced with the same amount of CP for four weeks. Rats fed CP had reduced plasma total homocysteine level and markedly increased levels of the choline pathway metabolites betaine, dimethylglycine, sarcosine, glycine and serine, as well as the transsulfuration pathway metabolites cystathionine and cysteine. Hepatic mRNA level of enzymes involved in homocysteine remethylation, methionine synthase and betaine-homocysteine S-methyltransferase, were unchanged, whereas cystathionine gamma-lyase of the transsulfuration pathway was increased in the CP treated rats. Plasma concentrations of vitamin B2, folate, cobalamin, and the B-6 catabolite pyridoxic acid were increased in the 20% CP-treated rats. In conclusion, the CP diet was associated with lower plasma homocysteine concentration and higher levels of serine, choline oxidation and transsulfuration metabolites compared to a casein diet. The status of related B-vitamins was also affected by CP.

  8. Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

    Science.gov (United States)

    Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj

    2016-07-01

    PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.

  9. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation

    OpenAIRE

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-01-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21–22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, di...

  10. Enzyme-Enhanced Extraction of Phenolic Compounds and Proteins from Flaxseed Meal

    OpenAIRE

    Bernardo Dias Ribeiro; Daniel Weingart Barreto; Maria Alice Zarur Coelho

    2013-01-01

    Flaxseed (Linum usitatissimum) meal, the main byproduct of the flaxseed oil extraction process, is composed mainly of proteins, mucilage, and phenolic compounds. The extraction methods of phenolics either commonly employed the use of mixed solvents (dioxane/ethanol, water/acetone, water/methanol, and water/ethanol) or are done with the aid of alkaline, acid, or enzymatic hydrolysis. This work aimed at the study of optimal conditions for a clean process, using renewable solvents and enzymes, f...

  11. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes.

    Science.gov (United States)

    Han, Kyungsook; Nepal, Chirag

    2007-05-01

    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.

  12. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves

    NARCIS (Netherlands)

    Zhang, C.; Sanders, J.P.M.; Xiao, T.T.; Bruins, M.E.

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further impr

  13. Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neuropathogenic Clostridium botulinum

    Science.gov (United States)

    An acetone-sodium dodecyl sulfate (SDS) disruption method was used for the extraction of cellular proteins from neurotoxigenic Clostridium botulinum. The amount of protein extracted per gram of dry weight and the protein profile as revealed by polyacrylamide gel electrophoresis (PAGE) was comparabl...

  14. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves

    NARCIS (Netherlands)

    Zhang, C.; Sanders, J.P.M.; Xiao, T.T.; Bruins, M.E.

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further

  15. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves

    NARCIS (Netherlands)

    Zhang, C.; Sanders, J.P.M.; Xiao, T.T.; Bruins, M.E.

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further impr

  16. Effects of ultrasound-assisted α-amylase degradation treatment with multiple modes on the extraction of rice protein.

    Science.gov (United States)

    Yang, Xue; Li, Yunliang; Li, Suyun; Oladejo, Ayobami Olayemi; Wang, Yucheng; Huang, Shanfen; Zhou, Cunshan; Ye, Xiaofei; Ma, Haile; Duan, Yuqing

    2018-01-01

    The effects of ultrasound-assisted α-amylase degradation (UAD) treatment with multiple modes on the extraction of rice protein (RP) from rice dreg powder were studied. The results showed that UAD treatment increased protein extraction rate significantly (pextraction rate compared to that of simultaneous or single frequency ultrasound treatment. The highest protein extraction rate was obtained under 20/35kHz of sequential frequency UAD treatment with the protein extraction rate of 89.58% and protein purity of 92.99%. The test and analysis of fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, starch content, non-protein nitrogen content, SH and SS content, surface hydrophobicity, amino acids content and proteins characteristics were studied to reveal the mechanism of UAD on the protein extraction effect. After treated with UAD, the starch-protein agglomerates disintegrated completely, moreover, protein advanced structure was changed to some extent and the disulfide bond was partially broken, but the main chain structure (peptide chain), specificity of protein and amino acid composition were not obviously changed. Consequently, the structure modified RP extracted by UAD treatment presented higher solubility, emulsifying activity and foaming capacity, which was beneficial for the application of RP. In conclusion, UAD treatment is an effective method for maximizing extraction rate, purity and modifying properties of extracted protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae).

    Science.gov (United States)

    Chia, Mathias Ahii; Lombardi, Ana Teresa; da Graça Gama Melão, Maria; Parrish, Christopher C

    2015-03-01

    Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

  18. Fatty Acid Transport Protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    Science.gov (United States)

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-01-01

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC50 8–11μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC50 58μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of 13C-oleate demonstrating its potential as a therapeutic agent. PMID:26284975

  19. Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection.

    Science.gov (United States)

    Bergmann, C W; Ito, Y; Singer, D; Albersheim, P; Darvill, A G; Benhamou, N; Nuss, L; Salvi, G; Cervone, F; De Lorenzo, G

    1994-05-01

    Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.

  20. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    Science.gov (United States)

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  1. An Improvement on the Protein Extraction Procedure from Eggshell Membrane

    Institute of Scientific and Technical Information of China (English)

    Peng Ruo-ting; Guo Yulong

    2010-01-01

    @@ 1 Introduction Collagen is a frequently occurring protein found in poultry,livestock and human body,esp.in the hair,skin,bone cartilage,tendon and so on.It forms the strongly insoluble fibers and serves as connective tissue supporting materials.Nowadays it became one of the most important raw materials of bio-engineering materials which are widely used in various fields including cosmetics,biomedical and foods industries.With the outburst of multiple diseases from cows and pigs such as bovine spongiform encephalopathy(BSE),related authorities in different countries have estabhshed many restrictions on trading collagen from mammalians.Due to this ground scientists began to identify new resources of collagens.

  2. A tri-gram based feature extraction technique using linear probabilities of position specific scoring matrix for protein fold recognition.

    Science.gov (United States)

    Paliwal, Kuldip K; Sharma, Alok; Lyons, James; Dehzangi, Abdollah

    2014-03-01

    In biological sciences, the deciphering of a three dimensional structure of a protein sequence is considered to be an important and challenging task. The identification of protein folds from primary protein sequences is an intermediate step in discovering the three dimensional structure of a protein. This can be done by utilizing feature extraction technique to accurately extract all the relevant information followed by employing a suitable classifier to label an unknown protein. In the past, several feature extraction techniques have been developed but with limited recognition accuracy only. In this study, we have developed a feature extraction technique based on tri-grams computed directly from Position Specific Scoring Matrices. The effectiveness of the feature extraction technique has been shown on two benchmark datasets. The proposed technique exhibits up to 4.4% improvement in protein fold recognition accuracy compared to the state-of-the-art feature extraction techniques.

  3. An extraordinary accumulation of (-)-pinoresinol in cell-free extracts of Forsythia intermedia: evidence for enantiospecific reduction of (+)-pinoresinol.

    Science.gov (United States)

    Katayama, T; Davin, L B; Lewis, N G

    1992-11-01

    Stereoselective and enantiospecific transformation mechanisms in lignan biogenesis are only now yielding to scientific inquiry: it has been shown that soluble cell-free preparations from Forsythia intermedia catalyse the formation of the enantiomerically pure lignan, (-)-secoisolariciresinol, when incubated with coniferyl alcohol in the presence of NAD(P)H and H2O2. Surprisingly, (-)-pinoresinol also accumulates in this soluble cell-free assay mixture in > 96% enantiomeric excess, even though it is not the naturally occurring antipode present in Forsythia sp. But these soluble cell-free preparations do not engender stereoselective coupling; instead, racemic pinoresinols are first formed, catalysed by an H2O2-dependent peroxidase reaction. An enantiospecific NAD(P)H reductase then converts (+)-pinoresinol, and not the (-)-antipode, into (-)-secoisolariciresinol. Stereoselective synthesis [correction of syntheis] of (+)-pinoresinol from E-coniferyl alcohol is, however, catalysed by an insoluble enzyme preparation in F. suspensa, obtained following removal of readily soluble and ionically bound enzymes; no exogenously supplied cofactors were required other than oxygen, although the reaction was stimulated by NAD-malate addition. Thus, the overall biochemical pathway to enantiomerically pure (-)-secoisolariciresinol has been delineated.

  4. Rice proteins, extracted by alkali and α-amylase, differently affect in vitro antioxidant activity.

    Science.gov (United States)

    Wang, Zhengxuan; Liu, Ye; Li, Hui; Yang, Lin

    2016-09-01

    Alkali treatment and α-amylase degradation are different processes for rice protein (RP) isolation. The major aim of this study was to determine the influence of two different extraction methods on the antioxidant capacities of RPA, extracted by alkaline (0.2% NaOH), and RPE, extracted by α-amylase, during in vitro digestion for 2h with pepsin and for 3h with pancreatin. Upon pepsin-pancreatin digestion, the protein hydrolysates (RPA-S, RPE-S), which were the supernatants in the absence of undigested residue, and the whole protein digests (RPA, RPE), in which undigested residue remained, were measured. RPE exhibited the stronger antioxidant responses to free radical scavenging activity, metal chelating activity, and reducing power, whereas the weakest antioxidant capacities were produced by RPE-S. In contrast, no significant differences in antioxidant activity were observed between RPA and RPA-S. The present study demonstrated that the in vitro antioxidant responses induced by the hydrolysates and the protein digests of RPs could be affected differently by alkali treatment and α-amylase degradation, suggesting that the extraction is a vital processing step to modify the antioxidant capacities of RPs. The results of the current study indicated that the protein digests, in which undigested residues remained, could exhibit more efficacious antioxidant activity compared to the hydrolysates.

  5. Comparative extraction of erythrocyte EDTA-membrane proteins by some ionic and non-ionic detergents.

    Science.gov (United States)

    Ballestrin, G; Covaz, L; Scutari, G

    1980-06-15

    In order to examine whether it would be possible to obtain, by a simple extraction procedure from EDTA-erythrocyte-membranes, a partially purified preparation of the "band 3 zone" proteins, we have tested four solubilizing agents of common use. Detergents, both ionic (DOC and SDS) and non ionic (Tween 80 and Triton X-100), were not able, in our experimental conditions, to completely solubilize erythrocyte fragmented membranes which had previously been washed in EDTA-buffers. However, they were able to solubilize some of the membrane proteins, which could then be separated by SDS-PGE. The PGE densitometric profiles reported in this communication indicate that the protein mixture extracted by the ionic detergents DOC and SDS qualitatively reflects the protein composition of the membranes. Among the non ionic detergents, on the other hand, Triton X-100 appeared to be able to extract mainly one band (most probably the band 3 zone), while Tween 80 did not apparently extract any of the membrane proteins. Detergent concentrations, medium composition and experimental procedures are described in detail.

  6. A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomics Studies of Aphid Proteins

    Science.gov (United States)

    Few attempts have been made to methodically compare protein isolation methods from insect tissues for proteomic studies. To address this, we compared qualitative and quantitative differences among three methods for isolation, purification and solubilization of insect proteins. Schizaphis graminum,...

  7. Protein tyrosine phosphatase is possibly involved in cellular signal transduction and the regulation of ABA accumulation in response to water deficit in Maize L. coleoptile

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Water deficit-induced ABA accumulation is an ideal model or "stimulus-response" system to investigate cellular stress signaling in plant cells, using such a model the cellular stress signaling triggered by water deficit was investigated in Maize L. coleoptile. Water deficit-induced ABA accumulation was sensitively blocked by NaVO3, a potent inhibitor both to plasma membrane H+-ATPase (PM-H+- ATPase) and protein tyrosine phosphatase (PTPase). However, while PM- H+-ATPase activity was unaffected under water deficit and PM- H+-ATPase activator did not induce an ABA accumulation instead of water deficit, water deficit induced an increase in the protein phosphatase activity, and furthermore, ABA accumulation was inhibited by PAO, a specific inhibitor of PTPase. These results indicate that protein phosphtases may be involved in the cellular signaling in response to water deficit. Further studies identified at least four species of protein phosphtase as assayed by using pNPP as substrate, among which one component was especially sensitive to NaVO3. The NaVO3-sensitive enzyme was purified and finally showed a protein band about 66 kD on SDS/PAGE. The purified enzyme showed a great activity to some specific PTPase substrates at pH 6.0. In addition to NaVO3, the enzyme was also sensitive to some other PTPase inhibitors such as Zn2+ and MO33+, but not to Ca2+ and Mg2+, indicating that it might be a protein tyrosine phosphatase. Interestingly, the purified enzyme could be deactivated by some reducing agent DTT, which was previously proved to be an inhibitor of water deficit-induced ABA accumulation. This result further proved that PTPase might be involved in the cellular signaling of ABA accumulation in response to water deficit.

  8. APOE epsilon 4 influences the pathological phenotype of Alzheimer's disease by favouring cerebrovascular over parenchymal accumulation of A beta protein.

    Science.gov (United States)

    Chalmers, K; Wilcock, G K; Love, S

    2003-06-01

    The relative amounts of amyloid beta-protein (A beta) in cerebral blood vessels and parenchyma vary considerably amongst patients with Alzheimer's disease (AD). Although several mechanisms have been proposed to explain this variability, the underlying genetic and environmental determinants are still unclear, as are the functional consequences. Polymorphisms in APOE, the gene for apolipoprotein E (ApoE), influence the risk of developing AD and of deposition of A beta within the brain. We examined the relationship between the APOE genotype and the relative extent of accumulation of A beta as plaques within the cerebral parenchyma and in cortical blood vessels in the form of cerebral amyloid angiopathy (CAA), in autopsy brain tissue from 125 AD cases and from 53 elderly, neurologically normal controls of which 19 had CAA without other neuropathological features of AD. In the AD cases, we also assessed whether the severity of CAA was related to the age of onset and duration of dementia, risk factors for atherosclerotic vascular disease, and histologically demonstrable cerebral infarcts or foci of haemorrhage. The APOE genotype was determined by a standard polymerase chain reaction-based method. Paraffin sections of frontal, temporal and parietal lobes were immunolabelled for A beta and the parenchymal A beta load (total A beta minus vessel-associated A beta) was quantified by computer-assisted image analysis. CAA severity was scored for cortical and leptomeningeal vessels. The relevant clinical data were obtained from the database of the South West Brain Bank. In AD, we found the severity of CAA to be strongly associated with the number of epsilon 4 alleles (P possession of the APOE epsilon 4 allele favours vascular over parenchymal accumulation of A beta in AD. This may influence the pathogenesis of neurodegeneration in epsilon 4-associated AD.

  9. A review on rice bran protein: its properties and extraction methods.

    Science.gov (United States)

    Fabian, Cynthia; Ju, Yi-Hsu

    2011-01-01

    Rice bran protein has been found to be of high quality and of importance for food and pharmaceutical applications. It is a plant protein that can be derived from rice bran, an abundant and cheap agricultural byproduct. The protein content in rice bran is about 10-15% and it consists of 37% water-soluble, 31% salt-soluble, 2% alcohol-soluble, and 27% alkali-soluble storage proteins. Its unique property as being hypoallergenic and having anti-cancer activity makes it a superior cereal protein that may find a wide range of applications. There were already reports on the extraction of rice bran protein several decades ago. However, as of now, commercial rice bran protein is still unavailable in the market. This review is aimed at providing valuable discussions on rice bran protein, that is, storage protein, its various properties, and extraction methods for the development of an effective processing scheme. Also, an update on the current processing methods is also included.

  10. Extraction and identification of membrane proteins from black widow spider eggs.

    Science.gov (United States)

    Fu, Si-Ling; Li, Jiang-Lin; Chen, Jia; Wang, Qiu-Ting; Li, Jian-Jun; Wang, Xian-Chun

    2015-07-18

    The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

  11. Identification of nuclear phosphatidylinositol 4,5-bisphosphate-interacting proteins by neomycin extraction.

    Science.gov (United States)

    Lewis, Aurélia E; Sommer, Lilly; Arntzen, Magnus Ø; Strahm, Yvan; Morrice, Nicholas A; Divecha, Nullin; D'Santos, Clive S

    2011-02-01

    Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.

  12. Effect of pomegranate peel extract on lipid and protein oxidation in beef meatballs during refrigerated storage.

    Science.gov (United States)

    Turgut, Sebahattin Serhat; Soyer, Ayla; Işıkçı, Fatma

    2016-06-01

    Antioxidant effect of pomegranate peel extract (PE) to retard lipid and protein oxidation was investigated in meatballs during refrigerated storage at 4±1°C. Concentrated lyophilised water extract of pomegranate peel was incorporated into freshly minced beef meat at 0.5% and 1% concentrations and compared with 0.01% butylated hydroxytoluene (BHT) as a reference and control (without any antioxidant). PE showed high phenolic content and antioxidant activity. In PE added samples, thiobarbituric acid reactive substances (TBARS) value, peroxide formation, loss of sulfhydryl groups and formation of protein carbonyls were lower than control (P<0.01) after 8 days of storage. Sensory evaluation with respect to colour and rancid odour revealed that PE incorporation in meatballs prolonged the refrigerated storage up to 8 days. Addition of both 0.5 and 1% PE in meatballs reduced lipid and protein oxidation and improved sensory scores. These results indicated that PE was effective on retarding lipid and protein oxidation.

  13. Relationship of Soluble Grape-Derived Proteins to Condensed Tannin Extractability during Red Wine Fermentation.

    Science.gov (United States)

    Springer, Lindsay F; Chen, Lei-An; Stahlecker, Avery C; Cousins, Peter; Sacks, Gavin L

    2016-11-02

    In red winemaking, the extractability of condensed tannins (CT) can vary considerably even under identical fermentation conditions, and several explanations for this phenomenon have been proposed. Recent work has demonstrated that grape pathogenesis-related proteins (PRPs) may limit retention of CT added to finished wines, but their relevance to CT extractability has not been evaluated. In this work, Vitis vinifera and interspecific hybrids (Vitis ssp.) from both hot and cool climates were vinified under small-scale, controlled conditions. The final CT concentration in wine was well modeled from initial grape tannin and juice protein concentrations using the Freundlich equation (r(2)= 0.686). In follow-up experiments, separation and pretreatment of juice by bentonite, heating, freezing, or exogenous tannin addition reduced protein concentrations in juices from two grape varieties. The bentonite treatment also led to greater wine CT for one of the varieties, indicating that prefermentation removal of grape protein may be a viable approach to increasing wine CT.

  14. Alkali-aided protein extraction of chicken dark meat: composition and stability to lipid oxidation of the recovered proteins.

    Science.gov (United States)

    Moayedi, V; Omana, D A; Chan, J; Xu, Y; Betti, M

    2010-04-01

    Chicken dark meat has been considered as a major underused commodity due to the increasing demand for further-processed breast meat products. One option to increase the utilization of chicken dark meat is to extract myofibrillar proteins and separate them from fat and pigments to enhance their application for the preparation of further-processed meat products. The objective of the current study was to determine the effect of pH, in the range of 10.5 to 12.0, on the alkaline solubilization process of chicken dark meat. Aspects studied were the effect of the alkali-aided process on protein content, lipid composition, lipid oxidation, and color characteristics of the extracted meat. Each experiment and each assay were done at least in triplicate. Lipid content of the extracted meat showed a 50% reduction compared with the chicken dark meat. Neutral lipids were reduced by 61.51%, whereas polar lipids were not affected by the alkali treatments. There was a higher amount of TBA reactive substances observed in the extracted meat compared with chicken dark meat, indicating that extracted meat was more susceptible to oxidation. Long-chain polyunsaturated fatty acids (22:4n-6, 20:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3), which were detected only in the polar lipids, were responsible for increasing lipid oxidation susceptibility of extracted meat compared with chicken dark meat. Alkali-aided extraction of chicken dark meat lightened the color of the meat. The redness, yellowness, and total heme pigments in extracted meat significantly decreased by 83, 11, and 53%, respectively, compared with chicken dark meat. Even though this process did not remove polar lipids, based on our early findings, the extracted meat had considerable physicochemical and textural properties for product preparation compared with those of raw dark meat. Hence, alkali recovery of protein can be considered a potentially useful method to increase the utilization of dark chicken meat.

  15. EFFECT OF TEMPERATURE AND PARTICLE SIZE ON THE ALKALINE EXTRACTION OF PROTEIN FROM CHICKEN BONE WASTE

    Directory of Open Access Journals (Sweden)

    Andri Cahyo Kumoro

    2012-02-01

    Full Text Available Chicken bone is a waste of chicken meat processing industry and restaurants that has not been used widely, even though it contains valuable organic compounds that are functionals, such as collagenous and non collagenous protein. This research was conducted to investigate the effect of temperature and particle size on the protein extraction from chicken bones using dilute sodium hydroxide solution. Controlled parameters in this study were the solvent in the form of sodium hydroxide solution, extraction time for 1 hour, pH 10.5, the ratio of chicken bone powder: solvent (1:4 w/v, and stirring speed 200 rpm. While the operating variables included the extraction temperature of 30oC, 55oC, and 80oC, and particle size of 150 and 250 μm. Experiments were carried out by heating of 300 mL of sodium hydroxide solution with pH 10.5 in a three-necked flask equipped with Leibig condenser, thermometer, mechanical agitator and sampling device to reach the desired temperature (30oC, 55oC, and 80oC. Then, a total of 75 g of chicken bone powders with desired particle size (150μm and 250μm was introduced into the sodium hydroxide solution and the stirrer was operated at speed of 200rpm. At every 10 minutes interval, as much as 10 mL samples were withdrawn for total protein analysis using Lowry-Folin method. The experiment was terminated after 1 hour. The results show that both increase in temperature and particle size caused an increase in the amount of extracted protein. Highest concentration of protein extracted was achieved at 630.99 mg/L, when the extraction was carried out using 250mm bone particles and temperature 80oC.

  16. Extraction, composition, and functional properties of dried alfalfa (Medicago sativa L.) leaf protein.

    Science.gov (United States)

    Hojilla-Evangelista, Mila P; Selling, Gordon W; Hatfield, Ronald; Digman, Matthew

    2017-02-01

    Alfalfa is considered a potential feedstock for biofuels; co-products with value-added uses would enhance process viability. This work evaluated dried alfalfa leaves for protein production and describes the functional properties of the protein. Dried alfalfa leaves contained 260 g kg(-1) dry basis (DB) crude protein, with albumins being the major fraction (260 g kg(-1) of total protein). Alkali solubilization for 2 h at 50 °C, acid precipitation, dialysis, and freeze-drying produced a protein concentrate (600 g kg(-1) DB crude protein). Alfalfa leaf protein concentrate showed moderate solubility (maximum 500 g kg(-1) soluble protein from pH 5.5 to 10), excellent emulsifying properties (activity 158-219 m(2)  g(-1) protein, stability 17-49 min) and minimal loss of solubility during heating at pH ≥ 7.0. It is technically feasible to extract protein with desirable emulsifying and heat stability properties from dried alfalfa leaves; however, the dried form may not be a practical starting material for protein production, given the difficulty of achieving high yields and high-purity protein product. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  17. Role for the A domain of unprocessed accumulation-associated protein (Aap) in the attachment phase of the Staphylococcus epidermidis biofilm phenotype.

    Science.gov (United States)

    Conlon, Brian P; Geoghegan, Joan A; Waters, Elaine M; McCarthy, Hannah; Rowe, Sarah E; Davies, Julia R; Schaeffer, Carolyn R; Foster, Timothy J; Fey, Paul D; O'Gara, James P

    2014-12-01

    The polysaccharide intercellular adhesin or the cell wall-anchored accumulation-associated protein (Aap) mediates cellular accumulation during Staphylococcus epidermidis biofilm maturation. Mutation of sortase, which anchors up to 11 proteins (including Aap) to the cell wall, blocked biofilm development by the cerebrospinal fluid isolate CSF41498. Aap was implicated in this phenotype when Western blots and two-dimensional (2D) electrophoresis revealed increased levels of the protein in culture supernatants. Unexpectedly, reduced levels of primary attachment were associated with impaired biofilm formation by CSF41498 srtA and aap mutants. In contrast to previous studies, which implicated Aap proteolytic cleavage and, specifically, the Aap B domains in biofilm accumulation, the CSF41498 Aap protein was unprocessed. Furthermore, aap appeared to play a less important role in the biofilm phenotype of S. epidermidis 1457, in which the Aap protein is processed. Anti-Aap A-domain IgG inhibited primary attachment and biofilm formation in strain CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains CSF41498 and 1457 were identical, implicating altered protease activity in the differential Aap processing results in the two strains. These data reveal a new role for the A domain of unprocessed Aap in the attachment phase of biofilm formation and suggest that extracellular protease activity can influence whether Aap contributes to the attachment or accumulation phases of the S. epidermidis biofilm phenotype.

  18. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    Science.gov (United States)

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

  19. Radiobiological long-term accumulation of environmental alpha radioactivity in extracted human teeth and animal bones in Malaysia.

    Science.gov (United States)

    Almayahi, B A; Tajuddin, A A; Jaafar, M S

    2014-03-01

    In this study, the radiobiological analysis of natural alpha emitters in extracted human teeth and animal bones from Malaysia was estimated. The microdistributions of alpha particles in tooth and bone samples were measured using CR-39 alpha-particle track detectors. The lowest and highest alpha emission rates in teeth in the Kedah and Perak states were 0.0080 ± 0.0005 mBq cm(-2) and 0.061 ± 0.008 mBq cm(-2), whereas those of bones in the Perlis and Kedah states were 0.0140 ± 0.0001 mBq cm(-2) and 0.7700 ± 0.0282 mBq cm(-2), respectively. The average alpha emission rate in male teeth was 0.0209 ± 0.0008 mBq cm(-2), whereas that of female teeth was 0.0199 ± 0.0010 mBq cm(-2). The alpha emission rate in teeth is higher in smokers (0.0228 ± 0.0008 mBq cm(-2)) than in non-smokers (0.0179 ± 0.0008 mBq cm(-2)). Such difference was found statistically significant (p < 0.01).

  20. Protein cryoprotective activity of a cytosolic small heat shock protein that accumulates constitutively in chestnut stems and is up-regulated by low and high temperatures.

    Science.gov (United States)

    Lopez-Matas, Maria-Angeles; Nuñez, Paulina; Soto, Alvaro; Allona, Isabel; Casado, Rosa; Collada, Carmen; Guevara, Maria-Angeles; Aragoncillo, Cipriano; Gomez, Luis

    2004-04-01

    Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.

  1. Comparison between B CR sequential extraction and geo-accumulation method to evaluate metal mobility in sediments of Dongting Lake, Central China

    Institute of Scientific and Technical Information of China (English)

    YAO Zhigang

    2008-01-01

    The form in which a metal exists strongly influences its mobility and thus,the effects on the environment.Operational methods of speciation analysis,such as the use of sequential extraction procedures,are commonly applied.The Dongting Lake,the second largest fresh-water lake in China,contains three China wetlands of international importance,the East Dongting Lake,South Dongting Lake,and West Dongting Lake.In this work,an optimized BCR sequential extraction procedure was used to assess the environmental risk of Cd,Cr,Cu,Ni,Pb and Zn in contaminated sediment of the Dongting Lake.The procedure was evaluated by using a certified reference material,BCR701.The results of the partitioning study indicated that in the lake sediments,more easily mobilized forms(acid exchangeable) were predominant for Cd,particularly in the samples from the East Dongting Lake.In contrast,the largest amount of Pb was associated with the iron and manganese oxide fractions and Cu,Zn,Cr,and Ni analyzed were mainly distributed in residual phase at an average percentage greater than 60% of the total metals.The potential risk to the lake's water contamination was highest in the East Dongting Lake based on the calculated contamination factors.On the other hand,the total metal content was determined as well by inductively coupled plasma and mass spectrometry(ICP-MS)and assessed by using geo-accumulation index.The assessment results using geo-accumulation index were compared with the information on metal speciation.Both were correspondent with each other.

  2. Boosting accumulation of neutral lipids in Rhodosporidium kratochvilovae HIMPA1 grown on hemp (Cannabis sativa Linn) seed aqueous extract as feedstock for biodiesel production.

    Science.gov (United States)

    Patel, Alok; Pravez, Mohammad; Deeba, Farha; Pruthi, Vikas; Singh, Rajesh P; Pruthi, Parul A

    2014-08-01

    Hemp seeds aqueous extract (HSAE) was used as cheap renewable feedstocks to grow novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 isolated from Himalayan permafrost soil. The yeast showed boosted triglyceride (TAG) accumulation in the lipid droplets (LDs) which were transesterified to biodiesel. The sonicated HSAE prepared lacked toxic inhibitors and showed enhanced total lipid content and lipid yield 55.56%, 8.39±0.57g/l in comparison to 41.92%, 6.2±0.8g/l from industrially used glucose synthetic medium, respectively. Supersized LDs (5.95±1.02μm) accumulated maximum TAG in sonicated HSAE grown cells were visualized by fluorescent BODIPY (505/515nm) stain. GC-MS analysis revealed unique longer carbon chain FAME profile containing Arachidic acid (C20:0) 5%, Behenic acid (C22:0) 9.7%, Heptacosanoic acid (C27:0) 14.98%, for the first time in this yeast when grown on industrially competent sonicated HSAE, showing more similarity to algal oils.

  3. In vivo effect of the lipido-sterolic extract of Serenoa repens (Permixon) on mast cell accumulation and glandular epithelium trophism in the rat prostate.

    Science.gov (United States)

    Mitropoulos, Dionisios; Kyroudi, Aspasia; Zervas, Anastasios; Papadoukakis, Stefanos; Giannopoulos, Aris; Kittas, Christos; Karayannacos, Panagiotis

    2002-04-01

    The Serenoa repens lipido-sterolic extract (SRLSE, Permixon, Pierre Fabre Medicament, Castres, France) is used to treat benign prostate hyperplasia. We studied the in vivo effect of SRLSE on mast cell accumulation and the histological characteristics of the rat ventral prostate. Adult Wistar rats received either tocopherol or SRLSE (50 and 100 mg/kg body weight, respectively) every second day for 90 days. Histological features were studied in hematoxylin-eosin stained tissue sections while mean mast cell numbers were determined in Giemsa-stained sections. The central region of the ventral prostate in treated animals showed significant changes with acinar epithelium becoming flat or low cuboidal. In the same region, mean mast cell number per optical field in the control, low-dose and high-dose groups were, respectively, 4.7+/-0.7, 3.4+/-1.0 and 2.4+/-0.6, showing a dose-dependent, statistically significant decrease. Administering SRLSE significantly reduces mast cell accumulation and provokes epithelium atrophy within the central area of the rat ventral prostate. These phenomena may participate in the clinical activity of the drug.

  4. Screening for carbohydrate-binding proteins in extracts of Uruguayan plants

    Directory of Open Access Journals (Sweden)

    Plá A.

    2003-01-01

    Full Text Available The presence of carbohydrate-binding proteins, namely lectins, ß-galactosidases and amylases, was determined in aqueous extracts of plants collected in Uruguay. Twenty-six extracts were prepared from 15 Uruguayan plants belonging to 12 Phanerogam families. Among them, 18 extracts caused hemagglutination (HAG that was inhibited by mono- and disaccharides in 13 cases, indicating the presence of lectins. The other 8 extracts did not cause any HAG with the four systems used to detect HAG activity (rabbit and mouse red cells, trypsin-treated rabbit and mouse red cells. For the extracts prepared from Solanum commersonii, HAG activity and HAG inhibition were similar for those prepared from tubers, leaves and fruits, with the chitocompounds being responsible for all the inhibitions. Purification of the S. commersonii tuber lectin was carried out by affinity chromatography on asialofetuin-Sepharose, and SDS-PAGE under reducing conditions gave a single band of Mr of approximately 80 kDa. The monomer N-acetylglucosamine did not inhibit HAG induced by the purified lectin, but chitobiose inhibited HAG at 24 mM and chitotriose inhibited it at 1 mM. ß-Galactosidase activity was detected in leaves and stems of Cayaponia martiana, and in seeds from Datura ferox. Only traces of amylase activity were detected in some of the extracts analyzed. The present screening increases knowledge about the occurrence of carbohydrate-binding proteins present in regional plants.

  5. Improvement of protein extraction from sunflower meal by hydrolysis with alcalase

    Directory of Open Access Journals (Sweden)

    Vioque, J.

    2003-12-01

    Full Text Available Extraction of proteins from defatted sunflower meal has been improved by addition of the protease alcalase during alkaline extraction. This method offers several additional advantages as compared to the traditional alkaline extraction without alcalase, which is usually carried out after a sedimentation/flotation step to remove the lignocellulosic fraction. As compared to extraction without alcalase, addition of 0.1% (v/v alcalase improved the yield of protein extraction from 57.5% to 87.4%, providing an extract that is 22% hydrolyzed. In addition, an increment of up to 4.5 times in protein solubility at low pH values is achieved, which correlates with the degree of hydrolysis. The extracts that were obtained in the presence of alcalase had a higher proline and glycine content, suggesting that the protease improves extraction of proline-rich and glycine-rich cell wall proteins that are part of the lignocellulosic fraction. These protein extracts can be directly dried without generation of wastewater, and the resulting fiber-rich material could be used for animal feeding.Se ha mejorado la extracción proteica de la harina desengrasa de girasol mediante la adición de la proteasa alcalasa durante la extracción alcalina. Este método ofrece varias ventajas adicionales en comparación con la extracción alcalina tradicional sin alcalasa, que se desarrolla normalmente mediante un proceso de flotación/sedimentación para retirar la fracción lignocelulósica. En comparación a la extracción sin alcalasa, la adicción de 0.1% (v/v de alcalasa mejora los rendimientos de extracción proteica desde un 57.5% a un 87.4%, dando un extracto con un 22% de grado de hidrólisis. Además se obtiene un incremento de hasta 4.5 veces de la solubilidad proteica a bajos pHs, que se correlaciona con el grado de hidrólisis. Los extractos obtenidos con alcalasa tenían un mayor contenido de prolina y glicina, sugiriendo que la proteasa mejora la extracción de las

  6. Heat Shock Protein Augmentation of Root Hot Water Extract on Adipogenic Differentiation in Murine 3T3-L1 Preadipocytes

    Directory of Open Access Journals (Sweden)

    Wenchie Marie L. Lumbera

    2016-03-01

    Full Text Available There is a high association of heat shock on the alteration of energy and lipid metabolism. The alterations associated with thermal stress are composed of gene expression changes and adaptation through biochemical responses. Previous study showed that Angelica gigas Nakai (AGN root extract promoted adipogenic differentiation in murine 3T3-L1 preadipocytes under the normal temperature condition. However, its effect in heat shocked 3T3-L1 cells has not been established. In this study, we investigated the effect of AGN root hot water extract in the adipogenic differentiation of murine 3T3-L1 preadipocytes following heat shock and its possible mechanism of action. Thermal stress procedure was executed within the same stage of preadipocyte confluence (G0 through incubation at 42°C for one hour and then allowed to recover at normal incubation temperature of 37°C for another hour before AGN treatment for both cell viability assay and Oil Red O. Cell viability assay showed that AGN was able to dose dependently (0 to 400 μg/mL increase cell proliferation under normal incubation temperature and also was able to prevent cytotoxicity due to heat shock accompanied by cell proliferation. Confluent preadipocytes were subjected into heat shock procedure, recovery and then AGN treatment prior to stimulation with the differentiation solution. Heat shocked preadipocytes exhibited reduced differentiation as supported by decreased amount of lipid accumulation in Oil Red O staining and triglyceride measurement. However, those heat shocked preadipocytes that then were given AGN extract showed a dose dependent increase in lipid accumulation as shown by both evaluation procedures. In line with these results, real-time polymerase chain reaction (RT-PCR and Western blot analysis showed that AGN increased adipogenic differentiation by upregulating heat shock protection related genes and proteins together with the adipogenic markers. These findings imply the potential of

  7. Development of soft extraction method for structural characterization of boreal forest soil proteins with MALDI-TOF/MS

    Science.gov (United States)

    Kanerva, Sanna; Ketola, Raimo A.; Kitunen, Veikko; Smolander, Aino; Kotiaho, Tapio

    2010-05-01

    Nitrogen (N) is usually the nutrient restricting productivity in boreal forests. Forest soils contain a great amount of nitrogen, but only a small part of it is in mineral form. Most part of soil N is bound in the structures of different organic compounds such as proteins, peptides, amino acids and more stabilized, refractory compounds. Due to the fact that soil organic N has a very important role in soil nutrient cycling and in plant nutrition, there is a need for more detailed knowledge of its chemistry in soil. Conventional methods to extract and analyze soil organic N are usually very destructive for structures of higher molecular weight organic compounds, such as proteins. The aim of this study was to characterize proteins extracted from boreal forest soil by "soft" extraction methods in order to maintain their molecular structure. The organic layer (F) from birch forest floor containing 78% of organic matter was sieved, freeze dried, pulverized, and extracted with a citrate or phosphate buffer (pH 6 or 8). Sequential extraction with the citrate or phosphate buffer and an SDS buffer (pH 6.8), slightly modified from the method of Chen et al. (2009, Proteomics 9: 4970-4973), was also done. Proteins were purified from the soil extract by extraction with buffered phenol and precipitated with methanol + 0.1M ammonium acetate at -20°C. Characterization of proteins was performed with matrix assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/MS) and the concentration of total proteins was measured using Bradford's method. Bovine serum albumin (BSA) was used as a positive control in the extractions and as a standard protein in Bradford's method. Our results showed that sequential extraction increased the amount of extracted proteins compared to the extractions without the SDS-buffer; however, it must be noted that the use of SDS-buffer very probably increased denaturization of proteins. Purification of proteins from crude soil extracts

  8. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    Energy Technology Data Exchange (ETDEWEB)

    Li Shikuo; Shen Yuhua; Xie Anjian; Yu Xuerong; Zhang Xiuzhen; Yang Liangbao; Li Chuanhao [School of Chemistry and Chemical Engineering, Anhui University, Hefei 230039 (China)

    2007-10-10

    We describe the formation of amorphous selenium ({alpha}-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO{sub 3}{sup 2-}) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO{sub 3}{sup 2-} ions to Se{sup 0}, but also controls the nucleation and growth of Se{sup 0}, and even participates in the formation of {alpha}-Se/protein composites. The size and shell thickness of the {alpha}-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO{sub 3}{sup 2-} ions by Capsicum annuum L extract.

  9. Value Added Processing of Aflatoxin Contaminated Peanut Meal: Aflatoxin Sequestration During Protein Extraction

    Science.gov (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...

  10. Absorption spectral analysis of proteins and free amino acids in Pleurotus ostreatus fruiting body extracts

    Science.gov (United States)

    Kostyshyn, S.; Gorshynska, I.; Guminetsky, S. G.

    2002-02-01

    The paper deals with the results of spectrophotometric studies of the extracts of Pleurotus ostreatus fruiting bodies, grown in natural conditions in different habitats of Chernivtsy region, in the spectral interval of 215 - 340 nm. It is shown that the samples reveal considerable difference both in free amino acid content and reserved protein content of albumins, globulins, prolamins, glutelins.

  11. THE IDENTIFICATION AND CHARACTERIZATION OF AN IGE-INDUCING PROTEIN IN METARHIZIUM ANISOPLIAE EXTRACT

    Science.gov (United States)

    The Identification and Characterization of an IgE-Inducing Protein in Metarhizium anisopliae ExtractMarsha D.W. Ward1, Lisa B. Copeland1, Maura J. Donahue2, and Jody A. Shoemaker31ORD, NHEERL, US EPA, RTP, NC; 2Oak Ridge Institute for Science and Education, Cincinnati...

  12. Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

    DEFF Research Database (Denmark)

    Matos, T.; Senkbeil, Silja; Mendonça, A.

    2013-01-01

    technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low...

  13. Experiments on the extraction and photoconversion of chenopodium chlorophyll protein CP 668

    NARCIS (Netherlands)

    Terpstra, Willemke

    1966-01-01

    1. 1. The amount of extractable, photoconvertible, protein-chlorophyll complex CP 668 from Chenopodium album leaves was found to be very variable, depending on site and day of leaf collection. 2. 2. Photoconversion of CP 668 into a 740-nm absorbing form probably does not occur in intact leaves;

  14. Unveiling the potential of novel yeast protein extracts in white wines clarification and stabilization

    Directory of Open Access Journals (Sweden)

    Joana Pinto Fernandes

    2015-03-01

    Full Text Available Fining agents derived from animal and mineral sources are widely used to clarify and stabilize white wines. Nevertheless, health and environmental problems are being raised, concerning the allergenic and environmental impact of some of those fining products. In this study, our aim is to validate the potential of non-allergenic protein extracts, obtained from an alternative and safe source, naturally present in wine: oenological yeasts. Three untreated white wines were used in this work in order to evaluate the impact of these novel yeast protein extracts (YPE in terms of the wine clarification and stabilization improvement. Two separated fining trials were thus conducted at laboratory scale and the yeast alternatives were compared with reference fining agents, obtained from mineral, animal and vegetable origins. Our results indicate that yeast protein extracts were capable to promote (i brilliance/color improvement, (ii turbidity reduction (76-89% comparing with the untreated wines and (iii production of compact and homogeneous lees (44% smaller volume than obtained with bentonite. Additionally, after submitting wines to natural and forced oxidations, YPE treatments revealed (iv different forms of colloidal stabilization, by presenting comparable or superior effects when particularly compared to casein. Altogether, this study reveals that yeast protein extracts represent a promising alternative for white wine fining, derived from an entirely non-allergenic and sustainable origin.

  15. Extraction, composition, and functional properties of dried alfalfa (Medicago sativa L.) leaf protein

    Science.gov (United States)

    Alfalfa, traditionally used for animal feed, has attracted attention as a potential feedstock for biofuels and the viability of the process would be enhanced by co-products with value-added uses. This study describes extraction of protein from dried alfalfa leaves and the functional properties of th...

  16. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C., E-mail: cdirusso2@unl.edu

    2015-09-25

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC{sub 50} 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC{sub 50} 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of {sup 13}C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata.

  17. Extraction and purification methods in downstream processing of plant-based recombinant proteins.

    Science.gov (United States)

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

    2016-04-01

    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.

  18. A fluorescence spectroscopic study of a coagulating protein extracted from Moringa oleifera seeds.

    Science.gov (United States)

    Kwaambwa, H M; Maikokera, R

    2007-11-15

    The fluorescence studies of coagulating protein extracted from Moringa oleifera seeds have been studied using steady-state intrinsic fluorescence. The fluorescence spectra are dominated by tryptophan emission and the emission peak maximum (lambda(max)=343+ or -2nm) indicated that the tryptophan residue is not located in the hydrophobic core of the protein. Changes in solution pH affected the protein conformation as indicated by changes in the tryptophan fluorescence above pH 9 whereas the ionic strength had minimal effect. The exposure and environments of the tryptophan residue were determined using collisional quenchers.

  19. Two applications of information extraction to biological science journal articles: enzyme interactions and protein structures.

    Science.gov (United States)

    Humphreys, K; Demetriou, G; Gaizauskas, R

    2000-01-01

    Information extraction technology, as defined and developed through the U.S. DARPA Message Understanding Conferences (MUCs), has proved successful at extracting information primarily from newswire texts and primarily in domains concerned with human activity. In this paper we consider the application of this technology to the extraction of information from scientific journal papers in the area of molecular biology. In particular, we describe how an information extraction system designed to participate in the MUC exercises has been modified for two bioinformatics applications: EMPathIE, concerned with enzyme and metabolic pathways; and PASTA, concerned with protein structure. Progress to date provides convincing grounds for believing that IE techniques will deliver novel and effective ways for scientists to make use of the core literature which defines their disciplines.

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

    Directory of Open Access Journals (Sweden)

    Maycon Fagundes Teixeira Reis

    2016-06-01

    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.

  1. Ins(1,4,5)P{sub 3} facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jing [Medical Research Center, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Department of Dental Implantology, School of Stomatology, Tongji University, Shanghai 200072 (China); Ogata, Shigenori [Joint Laboratory for Frontier Medical Science, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Segawa, Masaru [Central Laboratory for Pathology and Morphology, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Usune, Sadaharu [Research Laboratory of Biodynamics, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Zhao, Yumei [Department of Pediatric Dentistry, School of Dentistry of Shanghai Tongji University, Shanghai 200072 (China); Katsuragi, Takeshi, E-mail: katsurag@fukuoka-u.ac.jp [Medical Research Center, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan)

    2010-07-02

    So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5 {mu}g) after 3 days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1 {mu}M inositol(1,4,5)trisphosphate (Ins(1,4,5)P{sub 3}) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca{sup 2+}-removal from ICM and by adding thapsigargin, a Ca{sup 2+}-ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P{sub 3} receptor antagonists. Administration of 500 {mu}M adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P{sub 3}.

  2. MALDI analysis of proteins after extraction from dissolvable ethylene glycol diacrylate cross-linked polyacrylamide gels.

    Science.gov (United States)

    Papasotiriou, Dimitrios G; Markoutsa, Stavroula; Gorka, Jan; Schleiff, Enrico; Karas, Michael; Meyer, Bjoern

    2013-09-01

    Although the extraction of intact proteins from polyacrylamide gels followed by mass spectrometric molecular mass determination has been shown to be efficient, there is room for alternative approaches. Our study evaluates ethylene glycol diacrylate, a cleavable cross-linking agent used for a new type of dissolvable gels. It attains an ester linkage that can be hydrolyzed in alkali conditions. The separation performance of the new gel system was tested by 1D and 2D SDS-PAGE using the outer chloroplast envelope of Pisum sativum as well as a soluble protein fraction of human lymphocytes, respectively. Gel spot staining (CBB), dissolving, and extracting were conducted using a custom-developed workflow. It includes protein extraction with an ammonia-SDS buffer followed by methanol treatment to remove acrylamide filaments. Necessary purification for MALDI-TOF analysis was implemented using methanol-chloroform precipitation and perfusion HPLC. Both cleaning procedures were applied to several standard proteins of different molecular weight as well as 'real' biological samples (8-75 kDa). The protein amounts, which had to be loaded on the gel to detect a peak in MALDI-TOF MS, were in the range of 0.1 to 5 μg, and the required amount increased with increasing mass.

  3. A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots

    Directory of Open Access Journals (Sweden)

    Elisete Pains Rodrigues

    2012-01-01

    Full Text Available Sample preparation is a critical step in two-dimensional gel electrophoresis (2-DE of plant tissues. Here we describe a phenol/SDS procedure that, although greatly simplified, produced well-resolved and reproducible 2-DE profiles of protein extracts from soybean [Glycine max (L. Merril] roots. Extractions were made in three replicates using both the original and simplified procedure. To evaluate the quality of the extracted proteins, ten spots were randomly selected and identified by mass spectrometry (MS. The 2-DE gels were equally well resolved, with no streaks or smears, and no significant differences were observed in protein yield, reproducibility, resolution or number of spots. Mass spectra of the ten selected spots were compared with database entries and allowed high-quality identification of proteins. The simplified protocol described here presents considerable savings of time and reagents without compromising the quality of 2-DE protein profiles and compatibility with MS analysis, and may facilitate the progress of proteomics studies of legume-rhizobia interactions.

  4. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Konuma, Tsuyoshi [Icahn School of Medicine at Mount Sinai, Department of Structural and Chemical Biology (United States); Harada, Erisa [Suntory Foundation for Life Sciences, Bioorganic Research Institute (Japan); Sugase, Kenji, E-mail: sugase@sunbor.or.jp, E-mail: sugase@moleng.kyoto-u.ac.jp [Kyoto University, Department of Molecular Engineering, Graduate School of Engineering (Japan)

    2015-12-15

    Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

  5. Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots.

    Science.gov (United States)

    Vilhena, Milca B; Franco, Mônica R; Schmidt, Daiana; Carvalho, Giselle; Azevedo, Ricardo A

    2015-09-01

    Proteomics is an outstanding area in science whose increasing application has advanced to distinct purposes. A crucial aspect to achieve a good proteome resolution is the establishment of a methodology that results in the best quality and wide range representation of total proteins. Another important aspect is that in many studies, limited amounts of tissue and total protein in the tissue to be studied are found, making difficult the analysis. In order to test different parameters, combinations using minimum amount of tissue with 4 protocols for protein extraction from tomato (Solanum lycopersicum L.) leaves and roots were evaluated with special attention to their capacity for removing interferents and achieving suitable resolution in bidimensional gel electrophoresis, as well as satisfactory protein yield. Evaluation of the extraction protocols revealed large protein yield differences obtained for each one. TCA/acetone was shown to be the most efficient protocol, which allowed detection of 211 spots for leaves and 336 for roots using 500 µg of leaf protein and 800 µg of root protein per gel.

  6. Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots

    Directory of Open Access Journals (Sweden)

    MILCA B. VILHENA

    2015-09-01

    Full Text Available Proteomics is an outstanding area in science whose increasing application has advanced to distinct purposes. A crucial aspect to achieve a good proteome resolution is the establishment of a methodology that results in the best quality and wide range representation of total proteins. Another important aspect is that in many studies, limited amounts of tissue and total protein in the tissue to be studied are found, making difficult the analysis. In order to test different parameters, combinations using minimum amount of tissue with 4 protocols for protein extraction from tomato (Solanum lycopersicum L. leaves and roots were evaluated with special attention to their capacity for removing interferents and achieving suitable resolution in bidimensional gel electrophoresis, as well as satisfactory protein yield. Evaluation of the extraction protocols revealed large protein yield differences obtained for each one. TCA/acetone was shown to be the most efficient protocol, which allowed detection of 211 spots for leaves and 336 for roots using 500 µg of leaf protein and 800 µg of root protein per gel.

  7. Preliminary study on total protein extraction methods from Enterococcus faecalis biofilm.

    Science.gov (United States)

    Chen, W; Liang, J; He, Z; Jiang, W

    2016-08-30

    Enterococcus faecalis is the major pathogen of post-endodontic disease and refractory periapical periodontitis, and recent research on this species has focused on its pathogenicity. E. faecalis most often causes disease in the form of a biofilm, and total protein expression shows a strong association with its virulence. Therefore, the purpose of our study was to explore different methods of extracting the total proteins of the E. faecalis (ATCC 33186 standard strain) biofilm. The total proteins in the biofilm were extracted using an ultrasonication method with varied parameters, including duration, amplitude setting, period, and duty cycle. After the optimal conditions of ultrasonication were determined based on the protein profile from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the total protein content in the biofilm was detected using the bicinchoninic acid assay, Bradford Coomassie brilliant blue assay, and Lowry assay, and the results were compared and analyzed. The parameters for the optimal conditions of ultrasonication were as follows: a processing duration of 2 min, amplitude setting of 20%, and ultrasonication period of 4 s at a 50% duty cycle. The total protein content was 2299.1 mg/dish when measured by the bicinchoninic assay, 3793.8 mg/dish when measured by the Bradford Coomassie brilliant blue assay, and 1858.0 mg/dish when measured by the Lowry assay. These results demonstrate that the Bradford Coomassie brilliant blue assay is a simple and feasible method for use in detecting the total protein content in a bacterial biofilm.

  8. Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.

    Science.gov (United States)

    Hayama, Tadashi; Kiyokawa, Ena; Yoshida, Hideyuki; Imakyure, Osamu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2016-08-15

    We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).

  9. Temporal resolution of misfolded prion protein transport, accumulation, glial activation, and neuronal death in the retinas of mice inoculated with scrapie

    Science.gov (United States)

    Currently, there is a lack of pathologic landmarks to describe the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between the transport of misfolded prion protein from the brain to the retina, the accumulation of PrPSc in the retina, the respon...

  10. The roles of protein and lipid in the accumulation and distribution of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in plants grown in biosolids-amended soils.

    Science.gov (United States)

    Wen, Bei; Wu, Yali; Zhang, Hongna; Liu, Yu; Hu, Xiaoyu; Huang, Honglin; Zhang, Shuzhen

    2016-09-01

    The roles of protein and lipid in the accumulation and distribution of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in seven species of plants from biosolids-amended soils were investigated. The PFOS and PFOA root concentration factors (Croot/Csoil) ranged from 1.37 to 4.68 and 1.69 to 10.3 (ng/groot)/(ng/gsoil), respectively, while the translocation factors (Cshoot/Croot) ranged from 0.055 to 0.16 and 0.093 to 1.8 (ng/gshoot)/(ng/groot), respectively. The PFOS and PFOA accumulations in roots correlated positively with root protein contents (P protein and inhibition effects of lipid on root uptake. The translocation factors correlated positively with the ratios between protein contents in shoots to those in roots (P protein on PFOS and PFOA translocation. This study is the first to reveal the different roles of protein and lipid in the accumulation and distribution of PFOS and PFOA in plants.

  11. A continuum of research projects to improve extraction of oil and proteins in oilseed plants

    Directory of Open Access Journals (Sweden)

    Miquel Martine

    2011-05-01

    Full Text Available A key challenge in the actual context of fossil sources rarefaction, global warming, and of increase of the world global population, is to promote the use of molecules derived from renewable sources such as plants. Among these molecules, lipids and proteins are targets of interest. Plant lipids from oilseeds are attractive substitutes to the use of fossil oil. Till the beginning of the 20th century, numerous products used in the daily life were derived from natural renewable products. For instance, plant oil was commonly used as fuel for vehicles and was entering in the composition of paintings, lubricants etc. Unfortunately, natural oils have been progressively replaced by cheaper fossil oil in the fabrication of these products. Nowadays, fossil oils are becoming increasingly expensive being a finite comodity. It is thus important to reduce our dependence from fossil oil and develop substitution industries. Oilseeds contain important amounts of proteins which are mainly used in feed. As several kilograms of plant protein are needed to obtain one kilogram of animal protein, the interest toward using plant protein in food is reinforced. The developments of the use of plant lipids, as well as proteins are a major stakes for the competitiveness of European agriculture and industry, as well as for sustainable development. Extraction of oil and proteins from rapeseed has a significant cost, in term of energy and solvent uses, and finally affects the ultimate quality of the products (protein digestibility. In order to quantitatively extract seed reserves under mild conditions, it will be necessary to limit the amount of energy needed, and avoid any use of solvents. Ideally, seeds should be processed in a bio refinery. In this paper, we will describe how oilseeds store their reserves, and roadblocks for improving actual oilseed extraction processes. A continuum of research projects aimed at answering targeted questions will be presented, with selected

  12. Continuous protein recovery from whey using liquid-solid circulating fluidized bed ion-exchange extraction.

    Science.gov (United States)

    Lan, Qingdao; Bassi, Amarjeet; Zhu, Jing-Xu Jesse; Margaritis, Argyrios

    2002-04-20

    A liquid-solid circulating fluidized bed (LSCFB) continuous ion-exchange extraction system has been investigated for total protein recovery from whey solutions under various operating conditions. The effectiveness of a dynamic seal was evaluated between the riser and the downcomer, and the best conditions for the establishment of this seal were established. Start-up studies indicated that the system is robust and stable. Under optimal conditions, a productivity of 8.2 g of total protein removed per hour per kilogram of resin was achieved with a protein removal efficiency of 78.4%. However, higher overall protein recovery of up to 90% was also achieved under other conditions, with lower protein concentration in the effluent and a lower overall productivity.

  13. A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples.

    Science.gov (United States)

    Araújo, J E; Oliveira, E; Otero-Glez, A; Santos Nores, J; Igrejas, G; Lodeiro, C; Capelo, J L; Santos, H M

    2014-02-01

    Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial experimental design (2(4)) was used to determine the effects of the extracting conditions in the efficiency of protein recovery from formalin-fixed kidney samples. The following variables were assessed: temperature of extraction, pH of extraction, composition of the extracting buffer and the use ultrasonic energy applied with probe. It is clearly demonstrated that when hating and ultrasonic energy are used in conjunction, a 7-fold increase (p protein extraction is obtained if compared to extracting conditions for which neither heating nor ultrasonic energy are used. The optimization study was done following the amount of protein extracted by UV (Nanodrop(®) technology, protein ABS at 280 nm) and by 1D SDS-PAGE. Extracts obtained with the optimized conditions were subjected to LC-MALDI MS/MS. A total of 112 proteins were identified.

  14. Electrophoretic Extraction and Proteomic Characterization of Proteins Buried in Marine Sediments

    Directory of Open Access Journals (Sweden)

    Eli K. Moore

    2014-10-01

    Full Text Available Proteins are the largest defined molecular component of marine organic nitrogen, and hydrolysable amino acids, the building blocks of proteins, are important components of particulate nitrogen in marine sediments. In oceanic systems, the largest contributors are phytoplankton proteins, which have been tracked from newly produced bloom material through the water column to surface sediments in the Bering Sea, but it is not known if proteins buried deeper in sediment systems can be identified with confidence. Electrophoretic gel protein extraction methods followed by proteomic mass spectrometry and database searching were used as the methodology to identify buried phytoplankton proteins in sediments from the 8–10 cm section of a Bering Sea sediment core. More peptides and proteins were identified using an SDS-PAGE tube gel than a standard 1D flat gel or digesting the sediment directly with trypsin. The majority of proteins identified correlated to the marine diatom, Thalassiosira pseudonana, rather than bacterial protein sequences, indicating an algal source not only dominates the input, but also the preserved protein fraction. Abundant RuBisCO and fucoxanthin chlorophyll a/c binding proteins were identified, supporting algal sources of these proteins and reinforcing the proposed mechanisms that might protect proteins for long time periods. Some preserved peptides were identified in unexpected gel molecular weight ranges, indicating that some structural changes or charge alteration influenced the mobility of these products during electrophoresis isolation. Identifying buried photosystem proteins suggests that algal particulate matter is a significant fraction of the preserved organic carbon and nitrogen pools in marine sediments.

  15. In vivo screening of five phytochemicals/extracts and a fungal immunomodulatory protein against colibacillosis in broilers

    NARCIS (Netherlands)

    Peek, H.W.; Halkes, S.B.A.; Tomassen, M.M.M.; Mes, J.J.; Landman, W.J.M.

    2013-01-01

    Five phytochemicals/extracts (an extract from Echinacea purpurea, a ß-glucan-rich extract from Shiitake, betaine [Betain™], curcumin from Curcuma longa [turmeric] powder, carvacrol and also a recombinant fungal immunomodulatory protein [FIP] from Ganoderma lucidum) cloned and expressed in

  16. In vivo screening of five phytochemicals/extracts and a fungal immunomodulatory protein against colibacillosis in broilers

    NARCIS (Netherlands)

    Peek, H.W.; Halkes, S.B.A.; Tomassen, M.M.M.; Mes, J.J.; Landman, W.J.M.

    2013-01-01

    Five phytochemicals/extracts (an extract from Echinacea purpurea, a ß-glucan-rich extract from Shiitake, betaine [Betain™], curcumin from Curcuma longa [turmeric] powder, carvacrol and also a recombinant fungal immunomodulatory protein [FIP] from Ganoderma lucidum) cloned and expressed in Escherichi

  17. Combining protein extraction and anaerobic digestion to produce feed, fuel and fertilizer from green biomass – An organic biorefinery concept

    DEFF Research Database (Denmark)

    Fernandez, Maria Santamaria; Salces, Beatriz Molinuevo; Lübeck, Mette

    Organically grown green biomass (red clover, clover grass) was investigated as a resource for organic feed and organic fertilizer by combination of proteins extraction and anaerobic digestion of the residues. Extraction of proteins from both crops revealed very favourable amino acid composition...

  18. Automated Device for Asynchronous Extraction of RNA, DNA, or Protein Biomarkers from Surrogate Patient Samples.

    Science.gov (United States)

    Bitting, Anna L; Bordelon, Hali; Baglia, Mark L; Davis, Keersten M; Creecy, Amy E; Short, Philip A; Albert, Laura E; Karhade, Aditya V; Wright, David W; Haselton, Frederick R; Adams, Nicholas M

    2016-12-01

    Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein). © 2015 Society for Laboratory Automation and Screening.

  19. The integral and extrinsic bioactive proteins in the aqueous extracted soybean oil bodies.

    Science.gov (United States)

    Zhao, Luping; Chen, Yeming; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

    2013-10-01

    Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman-Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.

  20. Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.

    Science.gov (United States)

    Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T

    2014-06-24

    Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue. Our laboratory has taken a mechanistic approach to developing improved protein extraction protocols, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions, then applying this approach to a model system called a "tissue surrogate", which is a gel formed by treating high concentrations of cytoplasmic proteins with formaldehyde, and finally FFPE mouse liver tissue. Our studies indicate that elevated pressure improves the recovery of proteins from FFPE tissue surrogates by hydrating and promoting solubilization of highly aggregated proteins allowing for the subsequent reversal (by hydrolysis) of formaldehyde-induced protein adducts and cross-links. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency and up to a 30-fold increase in the number of non-redundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of non-redundant proteins identified in the FFPE tissue was nearly identical to that of the corresponding frozen tissue.

  1. Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

    Directory of Open Access Journals (Sweden)

    Taruna Khurana

    Full Text Available German cockroach (GCr allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.Single chain fragment variable (scFv antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.Chicken scFv's generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv's recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

  2. Effect of Bovine Plasma Protein on Autolysis and Gelation of Protein Extracted from Giant Squid (Dosidicus gigas Mantle

    Directory of Open Access Journals (Sweden)

    Laura Raquel Marquez-Alvarez

    2015-01-01

    Full Text Available The effect of bovine plasma protein (BPP on the inhibition of autolytic activity and its effect on the gelling properties of a protein concentrate (PC obtained from jumbo squid (Dosidicus gigas mantle were investigated. Sols and gels were prepared from the PC by adding different amounts of BPP (0, 1, and 2%. Dynamic oscillatory measurements indicated that systems with 1% BPP had a higher elastic modulus (G′, in which hydrophobic interactions were favored. Concerning the technological and textural quality of the gels, BPP caused a greater water holding capacity (WHC, force, cohesiveness, and elasticity, probably due to improvement of the electrostatic and hydrophobic interactions during gel formation. Scanning electron microscopy (SEM allowed visualization of the formation of more rigid and ordered gels with less porosity when BPP was added. Therefore, the addition of BPP improved the gelling capacity of proteins extracted from giant squid.

  3. Rice Protein Extracted by Different Methods Affects Cholesterol Metabolism in Rats Due to Its Lower Digestibility

    Directory of Open Access Journals (Sweden)

    Hongbo Liu

    2011-11-01

    Full Text Available To elucidate whether the digestibility is responsible for the hypocholesterolemic action of rice protein, the effects of rice proteins extracted by alkali (RP-A and α-amylase (RP-E on cholesterol metabolism were investigated in 7-week-old male Wistar rats fed cholesterol-free diets for 3 weeks. The in vitro and in vivo digestibility was significantly reduced by RP-A and RP-E as compared to casein (CAS. The digestibility was lower in RP-E than that of RP-A. Compared with CAS, the significant cholesterol-lowering effects were observed in rats fed by RP-A and RP-E. Fecal excretion of bile acids was significantly stimulated by RP-E, but not by RP-A. The apparent cholesterol absorption was more effectively inhibited by RP-E than RP-A because more fecal neutral sterols were excreted in rats fed RP-E. There was a significant correlation between protein digestibility and cholesterol absorption (r = 0.8662, P < 0.01, resulting in a significant correlation between protein digestibility and plasma cholesterol level (r = 0.7357, P < 0.01 in this study. The present study demonstrates that the digestibility of rice protein affected by extraction method plays a major role in the modulation of cholesterol metabolism. Results suggest that the hypocholesterolemic action induced by rice protein with lower digestibility primarily contribute to the inhibition of cholesterol absorption.

  4. Alkaline Extraction of Cobia (Rachycentroncanadum Proteins: Physicochemical characteristics, functional and thermal properties

    Directory of Open Access Journals (Sweden)

    Sabrina de La Rocha

    2015-04-01

    Full Text Available Cobia (Rachycentroncanadum is an important emerging species in Brazilian mariculture. The aim of the study was to obtain and characterize a protein isolate from cobia muscle using chemical extraction process by alkaline solubilization and isoelectric precipitation of proteins. The extraction yield was 98.17g/100g protein on a dry basis. The highest solubility and water holding capacity (WHC of cobiaprotein isolate (CPI was obtained at pH 11and 21.9mL of water per gram of protein. The electrophoretic profiles revealed masses characteristic of myofibrillar proteins (myosin and actin. The main peaks identified by qualitative spectroscopy analysis of the infrared spectrawere characteristic of peptide bonds such as amide I and amide II. The highest fusion and degradation points of CPI were 259.1°C and 378°C, respectively. The results showed that the CPI has great biotechnological value in various industrial areas that require a product of high protein value.

  5. The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation

    Directory of Open Access Journals (Sweden)

    Marcel Stark

    2014-01-01

    Full Text Available The standardized Ginkgo biloba extract EGb 761 has well-described antioxidative activities and effects on different cytoprotective signaling pathways. Consequently, a potential use of EGb 761 in neurodegenerative diseases has been proposed. A common characteristic feature of a variety of such disorders is the pathologic formation of protein aggregates, suggesting a crucial role for protein homeostasis. In this study, we show that EGb 761 increased the catalytic activity of the proteasome and enhanced protein degradation in cultured cells. We further investigated this effect in a cellular model of Huntington’s disease (HD by employing cells expressing pathologic variants of a polyglutamine protein (polyQ protein. We show that EGb 761 affected these cells by (i increasing proteasome activity and (ii inducing a more efficient degradation of aggregation-prone proteins. These results demonstrate a novel activity of EGb 761 on protein aggregates by enhancing proteasomal protein degradation, suggesting a therapeutic use in neurodegenerative disorders with a disturbed protein homeostasis.

  6. Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis.

    Science.gov (United States)

    Jiang, Xi-Wen; Wang, Jing; Chan, Leo Lai; Lam, Paul Kwan Sing; Gu, Ji-Dong

    2015-08-01

    Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.

  7. PPInterFinder--a mining tool for extracting causal relations on human proteins from literature.

    Science.gov (United States)

    Raja, Kalpana; Subramani, Suresh; Natarajan, Jeyakumar

    2013-01-01

    One of the most common and challenging problem in biomedical text mining is to mine protein-protein interactions (PPIs) from MEDLINE abstracts and full-text research articles because PPIs play a major role in understanding the various biological processes and the impact of proteins in diseases. We implemented, PPInterFinder--a web-based text mining tool to extract human PPIs from biomedical literature. PPInterFinder uses relation keyword co-occurrences with protein names to extract information on PPIs from MEDLINE abstracts and consists of three phases. First, it identifies the relation keyword using a parser with Tregex and a relation keyword dictionary. Next, it automatically identifies the candidate PPI pairs with a set of rules related to PPI recognition. Finally, it extracts the relations by matching the sentence with a set of 11 specific patterns based on the syntactic nature of PPI pair. We find that PPInterFinder is capable of predicting PPIs with the accuracy of 66.05% on AIMED corpus and outperforms most of the existing systems. DATABASE URL: http://www.biomining-bu.in/ppinterfinder/

  8. Protein from the fraction remaining after RNA extraction is useful for proteomics but care must be exercised in its application.

    Science.gov (United States)

    Yamaguchi, Hiromi; Hasegawa, Kiyoshi; Esumi, Mariko

    2013-08-01

    Simultaneous isolation of mRNA and proteins from a single small biopsy specimen can be useful for integrated omics studies. Here, we have improved the method for extracting protein from the fraction remaining after RNA isolation by TRIzol reagent, for application in protein and proteome analyses. Protein yield was reduced by half, but the patterns developed on 2D gels were equivalent to conventional urea extractions. Thus, although quantitative profiles of individual proteins were different from conventionally-isolated samples, overall profiles were similar. Therefore, this particular protein source is useful for proteomics but care must be exercised in its application.

  9. Accumulated SET protein up-regulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation.

    Science.gov (United States)

    Almeida, Luciana O; Garcia, Cristiana B; Matos-Silva, Flavia A; Curti, Carlos; Leopoldino, Andréia M

    2014-02-28

    SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET-hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.

  10. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

    Directory of Open Access Journals (Sweden)

    Xiaolin eWu

    2015-01-01

    Full Text Available ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5, deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs, late embryogenesis abundant (LEA proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed matu