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Sample records for extracted proteins accumulate

  1. Extraction of Proteins with ABS

    NARCIS (Netherlands)

    Desai, R.K.; Streefland, M.; Wijffels, R.H.; Eppink, M.H.M.

    2016-01-01

    Over the past years, there has been an increasing trend in research on the extraction and purification of proteins using aqueous biphasic systems (ABS) formed by polymers, e.g., polyethylene glycol (PEG). In general, when dealing with protein purification processes, it is essential to maintain their

  2. Utilization of aloe vera extract as electrolyte for an accumulator

    Science.gov (United States)

    Azmi, F.; Sispriatna, D.; Ikhsan, K.; Masrura, M.; Azzahra, S. S.; Mahidin; Supardan, M. D.

    2018-03-01

    Aloe vera contains acid, which has the potential to generate electric current. The objective of this research is to study the potency of aloe vera extract as electrolyte for an accumulator. Experimental results showed that aloe vera extract has no a stable value of voltage and currency. The voltage and currency of aloe vera extract were reduced more than 50% for 60 minutes. Then, aloe vera extract was mixed with accu zuur to produce electrolyte solution. The mixture composition of aloe vera extract to accu zuur of 50:50 (v/v) generated stable voltage and currency. The experimental results showed the potential use of aloe vera extract to reduce the chemicals used in a conventional electrolyte solution.

  3. Chronology of p53 protein accumulation in gastric carcinogenesis

    NARCIS (Netherlands)

    Craanen, M. E.; Blok, P.; Dekker, W.; Offerhaus, G. J.; Tytgat, G. N.

    1995-01-01

    p53 Protein accumulation in early gastric carcinoma was studied in relation to the histological type (Lauren classification) and the type of growth pattern, including the chronology of p53 protein accumulation during carcinogenesis. Forty five, paraffin embedded gastrectomy specimens from early

  4. Characteristics of retinol accumulation from serum retinol-binding protein by cultured sertoli cells

    International Nuclear Information System (INIS)

    Shingleton, J.L.; Skinner, M.K.; Ong, D.E.

    1989-01-01

    The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoil cells accumulated [ 3 H]retinol in a time- and temperature-dependent manner. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/μg of cellular DNA. Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [ 3 H]retinol-RBP for [ 3 H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [ 3 H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 μM. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. Competition studies indicated, however, that protein recognition is important in the retinol uptake process. RBP, CRBP, and CRBP(II) competed with [ 3 H]retinol-RBP for [ 3 H]retinol accumulation, but free retinol, retinol-bovine serum albumin, and retinol-β-lactoglobulin did not. These studies indicated that Sertoli cell uptake of retinol involved recognition of the retinol-RBP complex at the cell surface with subsequent internalization of retinol, but not RBP

  5. 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.

  6. 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...

  7. Extracting knowledge from protein structure geometry

    DEFF Research Database (Denmark)

    Røgen, Peter; Koehl, Patrice

    2013-01-01

    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. Dynein-Based Accumulation of Membranes Regulates Nuclear Expansion in Xenopus laevis Egg Extracts.

    Science.gov (United States)

    Hara, Yuki; Merten, Christoph A

    2015-06-08

    Nuclear size changes dynamically during development and has long been observed to correlate with the space surrounding the nucleus, as well as with the volume of the cell. Here we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter range, herein referred to as the nuclear domain. Under spatial constraints smaller than this nuclear domain, the size of microtubule-occupied space surrounding the nucleus turned out to be limiting for the accumulation of membranes around the nucleus via the motor protein dynein, therefore determining the speed of nuclear expansion. This mechanism explains how spatial information surrounding the nucleus, such as the positioning of the nucleus inside the cell, can control nuclear expansion. Copyright © 2015 Elsevier Inc. All rights reserved.

  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. Accumulation of some metals by legumes and their extractability from acid mine spoils

    International Nuclear Information System (INIS)

    Taylor, R.W.; Ibeabuchi, I.O.; Sistani, K.R.; Shuford, J.W.

    1992-01-01

    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 Lespedeza striata (Thung.) Hook and Arn, sericea lespedeza 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 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 > 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

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

    Directory of Open Access Journals (Sweden)

    Meijing Li

    2015-01-01

    Full Text Available 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 model, and PPI discovery model. The Protein-NER model, which is named ProNER, identifies the protein names based on two methods: dictionary-based method and machine learning-based method. ProNER is capable of identifying more proteins than dictionary-based Protein-NER model in other existing systems. The final discovered PPIs extracted via PPI discovery model are represented in detail because we showed the protein interaction types and the occurrence frequency through two different methods. In the experiments, the result shows that the performances achieved by our ProNER and PPI discovery model are better than other existing tools. PPIMiner applied this protein-named entity recognition approach and parsing tree based PPI extraction method to improve the performance of PPI extraction. We also provide an easy-to-use interface to access PPIs database and an online system for PPIs extraction and Protein-NER.

  12. ACCUMULATION OF RECOMBINANT FUSION PROTEIN – SECRETORY ANALOG OF Ag85B AND ESAT6 MYCOBACTERIUM TUBERCULOSIS PROTEINS – IN TRANSGENIC Lemna minor L. PLANTS

    Directory of Open Access Journals (Sweden)

    A.A.Peterson

    2015-10-01

    Full Text Available Determination of the presence of the recombinant fusion protein (ESAT6-Ag85B(ΔTMD-6His and its accumulation level in duckweed plants (Lemna minor L. was the aim of the research. ESAT6 and Ag85B are secretory proteins of Mycobacterium tuberculosis and are considered as potential candidates for development of new vaccine against tuberculosis (TB. Transgenic duckweed plants were obtained previously by Agrobacterium rhizogenes-mediated transformation and possessed fusion gene sequence esxA-fbpBΔTMD. Specific polyclonal antibodies were produced in immunized mice to identify levels of accumulation of TB antigens in plants. Recombinant antigen used for mice immunization was obtained in our laboratory by expression in E. coli. Western blot analysis revealed the recombinant tuberculosis antigen ESAT6-Ag85B(ΔTMD-6His in extracts from transgenic L. minor plants. The level of accumulation of the protein corresponds to 0.4-0.5 µg protein per 1 g of fresh weight of plant. Additionally, the accumulation of recombinant protein was investigated in lyophilized transgenic plants after 1.5 year storage. Duckweed plants accumulating a recombinant analogue of M. tuberculosis secretory proteins can be used for development of plant-based edible vaccines.

  13. Ocimum basilicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages.

    Science.gov (United States)

    Bravo, Elena; Amrani, Souliman; Aziz, Mohammed; Harnafi, Hicham; Napolitano, Mariarosaria

    2008-12-01

    Macrophage lipid accumulation induced by low density lipoproteins (LDL) plays a pivotal role in atherosclerotic plaque development. Previous work showed that Ocimum basilicum extract, used as hypocholesterolemic agent by traditional medicine in Morocco, has hypolipidemic activity in rat acute hyperlipimidemia. This study investigated the effects of ethanolic extract of O. basilicum on lipid accumulation in human macrophages. As modification of LDL increase atherogenicity of the particles we evaluated the effects of the extract on LDL oxidation. The extract caused a dose-related increase of LDL-resistance to Cu(2+)-induced oxidation. Furthermore, at the dose of 60 microg/ml, significantly decreases the accumulation of macrophage lipid droplets induced by modified LDL evaluated as by red-oil staining. Cholesterol esterification and triacylglycerol synthesis in the cells were not affected. Macrophage treatment with 60 microg/ml, but not 20 microg/ml, of the extract reduced newly synthesized unesterified cholesterol by about 60% and decreased scavenger receptors activity by about 20-30%, evaluated by the internalization of cholesterol carried by [(3)H]CE-aggregated-LDL. The results suggest that O. basilicum ethanolic extract has the capability to reduce foam cell formation through the reduction of cholesterol synthesis and the modulation of the activity of surface scavenger receptors.

  14. Fusarium proliferatum strains change fumonisin biosynthesis and accumulation when exposed to host plant extracts.

    Science.gov (United States)

    Górna, Karolina; Pawłowicz, Izabela; Waśkiewicz, Agnieszka; Stępień, Łukasz

    2016-01-01

    Fumonisin concentrations in mycelia and media were studied in liquid Fusarium proliferatum cultures supplemented with host plant extracts. Furthermore, the kinetics of fumonisin accumulation in media and mycelia collected before and after extract addition was analysed as well as the changes in the expression of the FUM1 gene. Fumonisin content in culture media increased in almost all F. proliferatum strains shortly after plant extracts were added. The asparagus extract induced the highest FB level increase and the garlic extract was the second most effective inducer. Fumonisin level decreased constantly until 14th day of culturing, though for some strains also at day 8th an elevated FB level was observed. Pineapple extract induced the highest increase of fum1 transcript levels as well as fumonisin synthesis in many strains, and the peas extract inhibited fungal growth and fumonisin biosynthesis. Moreover, fumonisins were accumulated in mycelia of studied strains and in the respective media. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  15. Curcumin and Curcuma longa L. extract ameliorate lipid accumulation through the regulation of the endoplasmic reticulum redox and ER stress.

    Science.gov (United States)

    Lee, Hwa-Young; Kim, Seung-Wook; Lee, Geum-Hwa; Choi, Min-Kyung; Chung, Han-Wool; Lee, Yong-Chul; Kim, Hyung-Ryong; Kwon, Ho Jeong; Chae, Han-Jung

    2017-07-26

    For this study, we examined the effects of curcumin against acute and chronic stress, paying specific attention to ROS. We also aimed to clarify the differences between acute and chronic stress conditions. We investigated the effects of curcumin against acute stress (once/1 day CCl 4 treatment) and chronic-stress (every other day/4week CCl 4 treatment). Compared with acute stress, in which the antioxidant system functioned properly and aspartate transaminase (AST) and ROS production increased, chronic stress increased AST, alanine aminotransferase (ALT), hepatic enzymes, and ROS more significantly, and the antioxidant system became impaired. We also found that ER-originated ROS accumulated in the chronic model, another difference between the two conditions. ER stress was induced consistently, and oxidative intra-ER protein folding status, representatively PDI, was impaired, especially in chronic stress. The PDI-associated client protein hepatic apoB accumulated with the PDI-binding status in chronic stress, and curcumin recovered the altered ER folding status, regulating ER stress and the resultant hepatic dyslipidemia. Throughout this study, curcumin and curcumin-rich Curcuma longa L. extract promoted recovery from CCl 4 -induced hepatic toxicity in both stress conditions. For both stress-associated hepatic dyslipidemia, curcumin and Curcuma longa L. extract might be recommendable to recover liver activity.

  16. Prediction of methylmercury accumulation in rice grains by chemical extraction methods

    International Nuclear Information System (INIS)

    Zhu, Dai-Wen; Zhong, Huan; Zeng, Qi-Long; Yin, Ying

    2015-01-01

    To explore the possibility of using chemical extraction methods to predict phytoavailability/bioaccumulation of soil-bound MeHg, MeHg extractions by three widely-used extractants (CaCl 2 , DTPA, and (NH 4 ) 2 S 2 O 3 ) were compared with MeHg accumulation in rice grains. Despite of variations in characteristics of different soils, MeHg extracted by (NH 4 ) 2 S 2 O 3 (highly affinitive to MeHg) correlated well with grain MeHg levels. Thus (NH 4 ) 2 S 2 O 3 extraction, solubilizing not only weakly-bound and but also strongly-bound MeHg, may provide a measure of ‘phytoavailable MeHg pool’ for rice plants. Besides, a better prediction of grain MeHg levels was obtained when growing condition of rice plants was also considered. However, MeHg extracted by CaCl 2 or DTPA, possibly quantifying ‘exchangeable MeHg pool’ or ‘weakly-complexed MeHg pool’ in soils, may not indicate phytoavailable MeHg or predict grain MeHg levels. Our results provided the possibility of predicting MeHg phytoavailability/bioaccumulation by (NH 4 ) 2 S 2 O 3 extraction, which could be useful in screening soils for rice cultivation in contaminated areas. - Highlights: • MeHg extraction by (NH 4 ) 2 S 2 O 3 correlates well with its accumulation in rice grains. • MeHg extraction by (NH 4 ) 2 S 2 O 3 provides a measure of phytoavailable MeHg in soils. • Some strongly-bound MeHg could be desorbed from soils and available to rice plants. • MeHg extraction by CaCl 2 or DTPA could not predict grain MeHg levels. - Methylmercury extraction from soils by (NH 4 ) 2 S 2 O 3 could possibly be used for predicting methylmercury phytoavailability and its bioaccumulation in rice grains

  17. 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)

  18. Tracking of pigment accumulation and secretion in extractive fermentation of Monascus anka GIM 3.592.

    Science.gov (United States)

    Chen, Gong; Bei, Qi; Huang, Tao; Wu, Zhenqiang

    2017-10-04

    Monascus pigments are promising sources for food and medicine due to their natural food-coloring functions and pharmaceutical values. The innovative technology of extractive fermentation is used to promote pigment productivity, but reports of pigment trans-membrane secretion mechanism are rare. In this study, tracking of pigment accumulation and secretion in extractive fermentation of Monascus anka GIM 3.592 was investigated. The increased vacuole size in mycelia correlated with fluorescence intensity (r > 0.85, p fermentation and batch fermentation, a threefold decrease in the NAD + /NADH ratio in mycelia and a more than 200-fold increase in glucose-6-phosphate dehydrogenase (G6PDH) activity in extracellular broth occurred, further suggesting that a reduction reaction for pigment conversion from orange pigments to yellow pigments occurred in non-aqueous phase solution. A putative model was established to track the localization of Monascus pigment accumulation and its trans-membrane secretion in extractive fermentation. This finding provides a theoretical explanation for microbial extractive fermentation of Monascus pigments, as well as other non-water-soluble products.

  19. 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. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development.

    Science.gov (United States)

    Marcon, Caroline; Schützenmeister, André; Schütz, Wolfgang; Madlung, Johannes; Piepho, Hans-Peter; Hochholdinger, Frank

    2010-12-03

    Heterosis describes the superior performance of heterozygous F(1)-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F(1)-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.

  1. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

    Science.gov (United States)

    Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

    2010-01-01

    Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

  2. 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. PMID:26840533

  3. 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.

  4. Studies on beam extraction from the 1 GeV proton accumulator ring

    International Nuclear Information System (INIS)

    Goyal, Pradeep Kumar; Sharma, Amalendu; Kumar, Vinit; Ghodke, A.D.

    2015-01-01

    For the proposed Indian Spallation Neutron Source (ISNS), a 1 GeV proton Accumulator Ring (AR) is presently being designed at RRCAT. Two optics configurations of AR, namely FODO and Hybrid lattices are under consideration. Each lattice configuration has four superperiods. In this paper, preliminary studies on beam extraction from AR are presented for both the optics configurations. The extraction system will be accommodated in one of the long dispersion free straight sections. Bunch length of the proton beam in AR is 700 ns, and the revolution time of the bunch in AR is 1 ms. This leaves a gap of ∼300 ns for bunch extraction. The proton bunch will be extracted to Ring to Target Beam Transport (RTBT) line, with the help of fast kicker and septum magnets. In this paper, we present the details of the beam extraction scheme with suitable number of kicker magnets, and find out their optimal location and strength. Estimation of field error tolerances for kicker magnets is also presented. (author)

  5. Body fat accumulation in zebrafish is induced by a diet rich in fat and reduced by supplementation with green tea extract.

    Directory of Open Access Journals (Sweden)

    Shinichi Meguro

    Full Text Available Fat-rich diets not only induce obesity in humans but also make animals obese. Therefore, animals that accumulate body fat in response to a high-fat diet (especially rodents are commonly used in obesity research. The effect of dietary fat on body fat accumulation is not fully understood in zebrafish, an excellent model of vertebrate lipid metabolism. Here, we explored the effects of dietary fat and green tea extract, which has anti-obesity properties, on body fat accumulation in zebrafish. Adult zebrafish were allocated to four diet groups and over 6 weeks were fed a high-fat diet containing basal diet plus two types of fat or a low-fat diet containing basal diet plus carbohydrate or protein. Another group of adult zebrafish was fed a high-fat diet with or without 5% green tea extract supplementation. Zebrafish fed the high-fat diets had nearly twice the body fat (visceral, subcutaneous, and total fat volume and body fat volume ratio (body fat volume/body weight of those fed low-fat diets. There were no differences in body fat accumulation between the two high-fat groups, nor were there any differences between the two low-fat groups. Adding green tea extract to the high-fat diet significantly suppressed body weight, body fat volume, and body fat volume ratio compared with the same diet lacking green tea extract. 3-Hydroxyacyl-coenzyme A dehydrogenase and citrate synthase activity in the liver and skeletal muscle were significantly higher in fish fed the diet supplemented with green tea extract than in those fed the unsupplemented diet. Our results suggest that a diet rich in fat, instead of protein or carbohydrate, induced body fat accumulation in zebrafish with mechanisms that might be similar to those in mammals. Consequently, zebrafish might serve as a good animal model for research into obesity induced by high-fat diets.

  6. Comparison of protein extraction methods suitable for proteomics ...

    African Journals Online (AJOL)

    An efficient protein extraction method is a prerequisite for successful implementation of proteomics. In this study, seedling roots of Jerusalem artichoke were treated with the concentration of 250 mM NaCl for 36 h. Subsequently, six different protocols of protein extraction were applied to seedling roots of Jerusalem artichoke ...

  7. Water Extract of Dolichos lablab Attenuates Hepatic Lipid Accumulation in a Cellular Nonalcoholic Fatty Liver Disease Model.

    Science.gov (United States)

    Im, A-Rang; Kim, Yun Hee; Lee, Hye Won; Song, Kwang Hoon

    2016-05-01

    Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that is rising in prevalence worldwide. Therapeutic strategies for patients with NAFLD are limited by a lack of effective drugs. In this report, we show that Dolichos lablab water extract (DLL-Ex) protects against free fatty acid (FFA)-induced lipid accumulation and attenuates expression of genes involved in lipid droplet accumulation in cellular NAFLD models. The hepatoprotective effects and underlying mechanism of DLL-Ex were assessed using an in vitro cellular model in which NAFLD was simulated by inducing excessive FFA influx into hepatocytes. HepG2 cells were treated with DLL-Ex and FFAs for 24 h, after which intracellular lipid content was observed by using Nile Red and Oil Red O staining. Quantitative real-time polymerase chain reaction was used to measure expression levels of genes related to FFA-mediated cellular energy depletion. Western blotting was used to measure protein levels of phosphorylated c-Jun N-terminal kinase, AMP-activated protein kinase alpha (AMPKα), and peroxisome proliferator-activated receptor γ coactivator 1 alpha. In HepG2 cells, DLL-Ex inhibited expression of CD36, which regulates fatty acid uptake, as well as BODIPY-labeled fatty acid uptake. Additionally, DLL-Ex significantly attenuated FFA-mediated cellular energy depletion and mitochondrial membrane depolarization. Furthermore, DLL-Ex enhanced phosphorylation of AMPK, indicating that AMPK is a critical regulator of DLL-Ex-mediated inhibition of hepatic lipid accumulation, possibly through its antioxidative effect. These results demonstrate that DLL-Ex exerts potent anti-NAFLD activity, suggesting that it could be a potential adjuvant treatment for patients with NAFLD.

  8. CONSTRUCTION AND POWER TEST OF THE EXTRACTION KICKER MAGNET FOR SNS ACCUMULATOR RING

    International Nuclear Information System (INIS)

    PAI, C.; HAHN, H.; HSEUH, H.; LEE, Y.; MENG, W.; MI, J.; SANDBERG, J.; TODD, R.

    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 OWL. 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

  9. Extractable protein content of radiation vulcanized natural rubber latex films

    International Nuclear Information System (INIS)

    Ma'zam Md Said; Wan Manshol Wan Zin

    1996-01-01

    The effects of processing conditions on extractable protein content of coagulant dipped radiation vulcanized natural rubber latex films have been investigated. Drying of wet-gel of radiation vulcanized latex films even at a relatively low temperature of 70 degree C resulted in increases of extractable protein content of the films. The extractable protein content is dependent upon both the temperature and time of drying of wet-gel deposit. Wet-gel leaching of film alone is not adequate to reduce the extractable protein content of films to low levels. Combination of wet-gel leaching, post-leaching, a dip in corn starch slurry, followed by drying at a low temperature of 70 degree C reduces the extractable protein content of films to very low levels

  10. 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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Extract from Edible Red Seaweed (Gelidium amansii) Inhibits Lipid Accumulation and ROS Production during Differentiation in 3T3-L1 Cells.

    Science.gov (United States)

    Seo, Min-Jung; Lee, Ok-Hwan; Choi, Hyeon-Son; Lee, Boo-Yong

    2012-06-01

    Gelidium (G.) amansii is a red alga widely distributed in the shallow waters around East Asian countries. We investigated the effect of G. amansii on lipid accumulation and ROS (Reactive Oxygen Species) production in 3T3-L1 cells. G. amansii extracts dose-dependently inhibited lipid formation and ROS generation in cultured cells. Our results showed that anti-adipogenic effect of G. amansii was due to the reduction in mRNA expressions of PPARγ peroxisome proliferator-activated receptor-γ and aP2 (adipocyte protein 2). G. amansii extracts significantly decreased mRNA levels of a ROS-generator, NOX4 (nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4), and increased the protein levels of antioxidant enzymes including SOD1/2 (superoxide dis-mutases), Gpx (glutathione peroxidase), and GR (glutathione reductase), which can lead to the reduction of ROS in the cell. In addition, the G. amansii extract enhanced mRNA levels of adiponectin, one of the adipokines secreted from adipocytes, and GLUT4, glucose uptake protein. Taken together, our study shows that G. amansii extract inhibited lipid accumulation and ROS production by controlling adipogenic signals and ROS regulating genes.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. Biogenesis of protein bodies during legumin accumulation in developing olive (Olea europaea L.) seed.

    Science.gov (United States)

    Jimenez-Lopez, Jose C; Zienkiewicz, Agnieszka; Zienkiewicz, Krzysztof; Alché, Juan D; Rodríguez-García, Maria I

    2016-03-01

    Much of our current knowledge about seed development and differentiation regarding reserves synthesis and accumulation come from monocot (cereals) plants. Studies in dicotyledonous seeds differentiation are limited to a few species and in oleaginous species are even scarcer despite their agronomic and economic importance. We examined the changes accompanying the differentiation of olive endosperm and cotyledon with a focus on protein bodies (PBs) biogenesis during legumin protein synthesis and accumulation, with the aim of getting insights and a better understanding of the PBs' formation process. Cotyledon and endosperm undergo differentiation during seed development, where an asynchronous time-course of protein synthesis, accumulation, and differential PB formation patterns was found in both tissues. At the end of seed maturation, a broad population of PBs, particularly in cotyledon cells, was distinguishable in terms of number per cell and morphometric and cytochemical features. Olive seed development is a tissue-dependent process characterized by differential rates of legumin accumulation and PB formation in the main tissues integrating seed. One of the main features of the impressive differentiation process is the specific formation of a broad group of PBs, particularly in cotyledon cells, which might depend on selective accumulation and packaging of proteins and specific polypeptides into PBs. The nature and availability of the major components detected in the PBs of olive seed are key parameters in order to consider the potential use of this material as a suitable source of carbon and nitrogen for animal or even human use.

  18. Comparison of protein extraction methods suitable for proteomics ...

    African Journals Online (AJOL)

    Jane

    2011-07-27

    Jul 27, 2011 ... An efficient protein extraction method is a prerequisite for successful implementation of proteomics. ... research, it is noteworthy to discover a proteome ..... Proteomic analysis of rice (Oryza sativa) seeds during germination.

  19. 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.

  20. Protein extractability from defatted Moringa oleifera lam. seeds flour ...

    African Journals Online (AJOL)

    Protein extractability from defatted Moringa oleifera seed flour was studied under various conditions of pH (2-10), time (5-60 minutes), salts (NaCl and CaCl ), salt concentrations (0-2 M) and solvent to flour ratio (10:1-30:1). 2 Results showed that protein extractability was dependent on pH, type of salt, salt concentrations and ...

  1. A charge-dependent mechanism is responsible for the dynamic accumulation of proteins inside nucleoli.

    Science.gov (United States)

    Musinova, Yana R; Kananykhina, Eugenia Y; Potashnikova, Daria M; Lisitsyna, Olga M; Sheval, Eugene V

    2015-01-01

    The majority of known nucleolar proteins are freely exchanged between the nucleolus and the surrounding nucleoplasm. One way proteins are retained in the nucleoli is by the presence of specific amino acid sequences, namely nucleolar localization signals (NoLSs). The mechanism by which NoLSs retain proteins inside the nucleoli is still unclear. Here, we present data showing that the charge-dependent (electrostatic) interactions of NoLSs with nucleolar components lead to nucleolar accumulation as follows: (i) known NoLSs are enriched in positively charged amino acids, but the NoLS structure is highly heterogeneous, and it is not possible to identify a consensus sequence for this type of signal; (ii) in two analyzed proteins (NF-κB-inducing kinase and HIV-1 Tat), the NoLS corresponds to a region that is enriched for positively charged amino acid residues; substituting charged amino acids with non-charged ones reduced the nucleolar accumulation in proportion to the charge reduction, and nucleolar accumulation efficiency was strongly correlated with the predicted charge of the tested sequences; and (iii) sequences containing only lysine or arginine residues (which were referred to as imitative NoLSs, or iNoLSs) are accumulated in the nucleoli in a charge-dependent manner. The results of experiments with iNoLSs suggested that charge-dependent accumulation inside the nucleoli was dependent on interactions with nucleolar RNAs. The results of this work are consistent with the hypothesis that nucleolar protein accumulation by NoLSs can be determined by the electrostatic interaction of positively charged regions with nucleolar RNAs rather than by any sequence-specific mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Optimized protein extraction for quantitative proteomics of yeasts.

    Directory of Open Access Journals (Sweden)

    Tobias von der Haar

    2007-10-01

    Full Text Available The absolute quantification of intracellular protein levels is technically demanding, but has recently become more prominent because novel approaches like systems biology and metabolic control analysis require knowledge of these parameters. Current protocols for the extraction of proteins from yeast cells are likely to introduce artifacts into quantification procedures because of incomplete or selective extraction.We have developed a novel procedure for protein extraction from S. cerevisiae based on chemical lysis and simultaneous solubilization in SDS and urea, which can extract the great majority of proteins to apparent completeness. The procedure can be used for different Saccharomyces yeast species and varying growth conditions, is suitable for high-throughput extraction in a 96-well format, and the resulting extracts can easily be post-processed for use in non-SDS compatible procedures like 2D gel electrophoresis.An improved method for quantitative protein extraction has been developed that removes some of the sources of artefacts in quantitative proteomics experiments, while at the same time allowing novel types of applications.

  3. Comparison of Protein Extracts from Various Unicellular Green Sources.

    Science.gov (United States)

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

    2017-09-13

    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.

  4. Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon.

    Science.gov (United States)

    Luks, Lisanne; Maier, Marcia Y; Sacchi, Silvia; Pollegioni, Loredano; Dietrich, Daniel R

    2017-11-01

    Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme D-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

  5. Defatting and Sonication Enhances Protein Extraction from Edible Insects.

    Science.gov (United States)

    Choi, Byoung Deug; Wong, Nathan A K; Auh, Joong-Hyuck

    2017-01-01

    Edible insects are attracting growing interest as a sustainable source of protein for addition to processed meat and dairy products. The current study investigated the optimal method for protein extraction from mealworm larvae ( Tenebrio molitor ), cricket adults ( Gryllus bimaculatus ), and silkworm pupae ( Bombyx mori ), for use in further applications. After defatting with n-hexane for up to 48 h, sonication was applied for 1-20 min and the protein yield was measured. All samples showed a total residual fat percentage below 1.36%, and a 35% to 94% improvement in protein yield (%). In conclusion, defatting with n-hexane combined with sonication improves the protein yield from insect samples.

  6. CHEMICAL CHARACTERIZATION OF A HYPOGLYCEMIC EXTRACT FROM CUCURBITA FICIFOLIA BOUCHE THAT INDUCES LIVER GLYCOGEN ACCUMULATION IN DIABETIC MICE.

    Science.gov (United States)

    Jessica, Garcia Gonzalez; Mario, Garcia Lorenzana; Alejandro, Zamilpa; Cesar, Almanza Perez Julio; Ivan, Jasso Villagomez E; Ruben, Roman Ramos; Javier, Alarcon-Aguilar Francisco

    2017-01-01

    The aqueous extract of Cucurbita ficifolia ( C. ficifolia ) fruit has demonstrated hypoglycemic effect, which may be attributed to some components in the extract. However, the major secondary metabolites in this fruit have not yet been identified and little is known about its extra-pancreatic action, in particular, on liver carbohydrate metabolism. Therefore, in addition to the isolation and structural elucidation of the principal components in the aqueous extract of C. ficifolia , the aim of this study was to determine whether or not the hypoglycemic effect of the aqueous extract of Cucurbita ficifolia ( C. ficifolia ) fruit is due to accumulation of liver glycogen in diabetic mice. The aqueous extract from fruit of C. ficifolia was fractionated and its main secondary metabolites were purified and chemically characterized (NMR and GC-MS). Alloxan-induced diabetic mice received daily by gavage the aqueous extract (30 days). The liver glycogen content was quantified by spectroscopic method and by PAS stain; ALT and AST by spectrometric method; glycogen synthase, glycogen phosphorylase and GLUT2 by Western blot; the mRNA expression of GLUT2 and glucagon-receptor by RT-PCR; while serum insulin was quantified by ELISA method. A liver histological analysis was also performed by H&E stain. Chemical fingerprint showed five majoritarian compounds in the aqueous extract of C. ficifolia : p -coumaric acid, p-hydroxybenzoic acid, salicin, stigmast-7,2,2-dien-3-ol and stigmast-7-en-3-ol. The histological analysis showed accumulation of liver glycogen. Also, increased glycogen synthase and decreased glycogen phosphorylase were observed. Interestingly, the histological architecture evidenced a liver-protective effect due the extract. Five compounds were identified in C. ficifolia aqueous extract. The hypoglycemic effect of this extract may be partially explained by liver glycogen accumulation. The bioactive compound responsible for the hypoglycemic effect of this extract will be

  7. Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

    Science.gov (United States)

    Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

    2010-02-01

    Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

  8. 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.

  9. Bone protein extraction without demineralization using principles from hydroxyapatite chromatography.

    Science.gov (United States)

    Cleland, Timothy P; Vashishth, Deepak

    2015-03-01

    Historically, extraction of bone proteins has relied on the use of demineralization to better retrieve proteins from the extracellular matrix; however, demineralization can be a slow process that restricts subsequent analysis of the samples. Here, we developed a novel protein extraction method that does not use demineralization but instead uses a methodology from hydroxyapatite chromatography where high concentrations of ammonium phosphate and ammonium bicarbonate are used to extract bone proteins. We report that this method has a higher yield than those with previously published small-scale extant bone extractions, with and without demineralization. Furthermore, after digestion with trypsin and subsequent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, we were able to detect several extracellular matrix and vascular proteins in addition to collagen I and osteocalcin. Our new method has the potential to isolate proteins within a short period (4h) and provide information about bone proteins that may be lost during demineralization or with the use of denaturing agents. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Extractable protein of radiation vulcanized natural rubber latex

    International Nuclear Information System (INIS)

    Soebianto, Y.S.; Upul, R.M.; Makuuchi, K.; Yoshii, F.; Kume, T.

    2000-01-01

    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)

  11. 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)

  12. 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.

  13. Organ accumulation and subcellular location of Cicer arietinum ST1 protein.

    Science.gov (United States)

    Albornos, Lucía; Cabrera, Javier; Hernández-Nistal, Josefina; Martín, Ignacio; Labrador, Emilia; Dopico, Berta

    2014-07-01

    The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. The movement protein and coat protein of alfalfa mosaic virus accumulate in structurally modified plasmodesmata

    NARCIS (Netherlands)

    van der Wel, N. N.; Goldbach, R. W.; van Lent, J. W.

    1998-01-01

    In systemically infected tissues of Nicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are

  15. 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...... was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins...... 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...

  16. Extractable protein of radiation vulcanized natural rubber latex

    International Nuclear Information System (INIS)

    Soebianto, Y.S.; Ratnayake, U.M.; Makuuchi, Keizo; Yoshii, Fumio; Kume, Tamikazu

    2000-01-01

    Protein remained in the latex products are reported to cause serious allergy. 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) in three different processes. The amount of WSP was 3 phr. It was only added to RVNRL (standard), added to re-centrifuged RVNRL (pre-centrifugation), and added to RVNRL followed by centrifugation (post-centrifugation). The protein content was determined by enhanced BCA method, and identified by SDS-PAGE. Extractable protein (EP) from the rubber has been reduced up to the minimum protein detection by combining WSP addition and centrifugation. Short leaching time (20-30 min.) can be achieved after the combine treatment, and SDS-PAGE 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 efficiency of protein removal by WSP depends on its molecular weight of WSP which relates to its water solubility. (author)

  17. Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases.

    Science.gov (United States)

    Bahr, Ben A; Wisniewski, Meagan L; Butler, David

    2012-04-01

    Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ(1-38) peptide corresponded with decreased levels of Aβ(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders to enhance

  18. How and why do toxic conformers of aberrant proteins accumulate during ageing?

    Science.gov (United States)

    Josefson, Rebecca; Andersson, Rebecca; Nyström, Thomas

    2017-07-15

    Ageing can be defined as a gradual decline in cellular and physical functions accompanied by an increased sensitivity to the environment and risk of death. The increased risk of mortality is causally connected to a gradual, intracellular accumulation of so-called ageing factors, of which damaged and aggregated proteins are believed to be one. Such aggregated proteins also contribute to several age-related neurodegenerative disorders e.g. Alzheimer's, Parkinson's, and Huntington's diseases, highlighting the importance of protein quality control (PQC) in ageing and its associated diseases. PQC consists of two interrelated systems: the temporal control system aimed at refolding, repairing, and/or removing aberrant proteins and their aggregates and the spatial control system aimed at harnessing the potential toxicity of aberrant proteins by sequestering them at specific cellular locations. The accumulation of toxic conformers of aberrant proteins during ageing is often declared to be a consequence of an incapacitated temporal PQC system-i.e. a gradual decline in the activity of chaperones and proteases. Here, we review the current knowledge on PQC in relation to ageing and highlight that the breakdown of both temporal and spatial PQC may contribute to ageing and thus comprise potential targets for therapeutic interventions of the ageing process. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  19. 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

  20. The correlation between heat-shock protein accumulation and persistence and chilling tolerance in tomato fruit

    International Nuclear Information System (INIS)

    Sabehat, A.; Weiss, D.; Lurie, S.

    1996-01-01

    Heating tomato fruit (Lycopersicon esculentum) for 48 h at 38 degrees C prevented chilling injury from developing after 21 d at 2 degrees C, whereas unheated fruit developed high levels of injury. Although the overall protein pattern as seen by Coomassie blue staining was similar from heated and unheated fruit, some high- and many low-molecular-mass proteins were observed in the heated fruit that were absent or present in reduced amounts in unheated fruit. When fruit were injected with [35S]methionine at harvest and then heated, they accumulated high levels of specific radiolabeled proteins that could still be detected after 21 d at 2 degrees C. If the fruit were held at 20 degrees C after heating, the label in the proteins declined rapidly and these fruit were also sensitive to chilling injury. Hsp70 antibody reacted more strongly with proteins from heated and chilled fruit than with proteins from chilled fruit. Hsp18.1 antibody reacted strongly with proteins from heated fruit but not with those from unheated fruit. A 23-kD protein, highly labeled in heated fruit but not in unheated fruit, had its amino terminus sequenced. To our knowledge, this is the first report showing a relationship between the persistence of heat-shock proteins and chilling tolerance in a plant tissue

  1. Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG)

    OpenAIRE

    Worby, Carolyn A.; Gentry, Matthew S.; Dixon, Jack E.

    2007-01-01

    Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual-specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PT...

  2. 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

  3. Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover

    International Nuclear Information System (INIS)

    Ju, H.-J.; Ye, C.-M.; Verchot-Lubicz, Jeanmarie

    2008-01-01

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion to accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocate to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway

  4. Adipocyte differentiation-related protein promotes lipid accumulation in goat mammary epithelial cells.

    Science.gov (United States)

    Shi, H B; Yu, K; Luo, J; Li, J; Tian, H B; Zhu, J J; Sun, Y T; Yao, D W; Xu, H F; Shi, H P; Loor, J J

    2015-10-01

    Milk fat originates from the secretion of cytosolic lipid droplets (CLD) synthesized within mammary epithelial cells. Adipocyte differentiation-related protein (ADRP; gene symbol PLIN2) is a CLD-binding protein that is crucial for synthesis of mature CLD. Our hypothesis was that ADRP regulates CLD production and metabolism in goat mammary epithelial cells (GMEC) and thus plays a role in determining milk fat content. To understand the role of ADRP in ruminant milk fat metabolism, ADRP (PLIN2) was overexpressed or knocked down in GMEC using an adenovirus system. Immunocytochemical staining revealed that ADRP localized to the surface of CLD. Supplementation with oleic acid (OA) enhanced its colocalization with CLD surface and enhanced lipid accumulation. Overexpression of ADRP increased lipid accumulation and the concentration of triacylglycerol in GMEC. In contrast, morphological examination revealed that knockdown of ADRP decreased lipid accumulation even when OA was supplemented. This response was confirmed by the reduction in mass of cellular TG when ADRP was knocked down. The fact that knockdown of ADRP did not completely eliminate lipid accumulation at a morphological level in GMEC without OA suggests that some other compensatory factors may also aid in the process of CLD formation. The ADRP reversed the decrease of CLD accumulation induced by adipose triglyceride lipase. This is highly suggestive of ADRP promoting triacylglycerol stability within CLD by preventing access to adipose triglyceride lipase. Collectively, these data provide direct in vitro evidence that ADRP plays a key role in CLD formation and stability in GMEC. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Antiproliferative activity of protein extracts from the black clam ...

    African Journals Online (AJOL)

    MB-231 cell lines with IC50 values of 138.035 and 157.19 μg/ml, respectively, and 2F2 showed IC50 values of 67.46 μg/ml on HeLa cells. These results suggest that protein extracts from C. fluctifraga might be potential anticancer agents.

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

    African Journals Online (AJOL)

    F. I. Abdullah

    2017-05-01

    May 1, 2017 ... extraction technique is highly relied on the protein structure and its chemical characteristics. ..... EDTA, 1% Triton X 100, 80% glycerol, 1M DTT and distilled water) and vortexed ..... prot5438, doi : 10.1101/pdb.prot5438.

  7. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. 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.

  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; Smit, John; Jiao, Yongqin

    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. The accumulation of a platelet protein, thrombospondin, at the site of arterial thrombus formation: Preliminary report

    International Nuclear Information System (INIS)

    Perlman, S.B.; Hammes, R.J.; Besozzi, M.C.; Folts, J.D.; Mosher, D.F.

    1987-01-01

    There are many methods available for the detection of thrombosis, none of which are noninvasive, rapid and accurate. Thrombosponding is a platelet protein that is present in the developing thrombus and may be an effective substance to use for imaging thrombosis. Vascular stenosis and thrombosis were produced in coronary, carotid and femoral arteries in eleven adult mongrel dogs. 131 I labeled thrombosponding was administered to each animal to determine whether the radiotracer accumulated at the site of thrombus formation. The radioactivity per gram of vessels with thrombi was significantly different from the control vessels or whole blood (p=0.0037 and p=0.015, respectively, paired t-test). This preliminary work suggests that iodinated thrombosponding accumulates at the site of thrombus formation. Labeled thrombosponding may be a rapid, safe and accurate method of detecting arterial thrombosis. (orig.)

  11. The extraction of proteins from the neem seed ( Indica azadirachta A ...

    African Journals Online (AJOL)

    Techniques for maximizing the extraction of protein from the neem seed (Indica azadirachta A. Juss) were investigated. Extractants used were sodium chloride and sodium sulphate solutions of varying concentration and pH. Maximum extractions of 17.86 g of extractable protein was obtained from 1 kg of crude protein, using ...

  12. Extraction and characterization of proteins from banana (Musa Sapientum L) flower and evaluation of antimicrobial activities.

    Science.gov (United States)

    Sitthiya, Kewalee; Devkota, Lavaraj; Sadiq, Muhammad Bilal; Anal, Anil Kumar

    2018-02-01

    Ultrasonic assisted alkaline extraction of protein from banana flower was optimized using response surface methodology. The extracted proteins were characterized by Fourier transform infrared spectroscopy and molecular weight distribution was determined by gel electrophoresis. The maximum protein yield of 252.25 mg/g was obtained under optimized extraction conditions: temperature 50 °C, 30 min extraction time and 1 M NaOH concentration. The alkaline extraction produced a significantly high protein yield compared to enzymatic extraction of banana flower. Chemical finger printing of proteins showed the presence of tyrosine, tryptophan and amide bonds in extracted protein. Alkaline and pepsin assisted extracted banana flower proteins showed characteristic bands at 40 and 10 kDA, respectively. The extracted proteins showed antibacterial effects against both gram positive and gram negative bacteria. The high protein content and antimicrobial activity indicate the potential applications of banana flower in the food and feed industry.

  13. 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

    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......-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...

  14. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    Science.gov (United States)

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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

  16. Accumulation and localization of extensin protein in apoplast of pea root nodule under aluminum stress.

    Science.gov (United States)

    Sujkowska-Rybkowska, Marzena; Borucki, Wojciech

    2014-12-01

    Cell wall components such as hydroxyproline-rich glycoproteins (HRGPs, extensins) have been proposed to be involved in aluminum (Al) resistance mechanisms in plants. We have characterized the distribution of extensin in pea (Pisum sativum L.) root nodules apoplast under short (for 2 and 24h) Al stress. Monoclonal antibodie LM1 have been used to locate extensin protein epitope by immunofluorescence and immunogold labeling. The nodules were shown to respond to Al stress by thickening of plant and infection thread (IT) walls and disturbances in threads growth and bacteria endocytosis. Immunoblot results indicated the presence of a 17-kDa band specific for LM1. Irrespective of the time of Al stress, extensin content increased in root nodules. Further observation utilizing fluorescence and transmission electron microscope showed that LM1 epitope was localized in walls and intercellular spaces of nodule cortex tissues and in the infection threads matrix. Al stress in nodules appears to be associated with higher extensin accumulation in matrix of enlarged thick-walled ITs. In addition to ITs, thickened walls and intercellular spaces of nodule cortex were also associated with intense extensin accumulation. These data suggest that Al-induced extensin accumulation in plant cell walls and ITs matrix may have influence on the process of IT growth and tissue and cell colonization by Rhizobium bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Olsen, H.; Wolfe, M.; Tell, J.; Tjeerdema, R.

    1995-01-01

    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

  18. Post-Transcriptional Regulation Prevents Accumulation of Glutathione Reductase Protein and Activity in the Bundle Sheath Cells of Maize1

    Science.gov (United States)

    Pastori, Gabriela M.; Mullineaux, Philip M.; Foyer, Christine H.

    2000-01-01

    Glutathione reductase (GR; EC 1.6.4.2) activity was assayed in bundle sheath and mesophyll cells of maize (Zea mays L. var H99) from plants grown at 20°C, 18°C, and 15°C. The purity of each fraction was determined by measuring the associated activity of the compartment-specific marker enzymes, Rubisco and phosphoenolpyruvate carboxylase, respectively. GR activity and the abundance of GR protein and mRNA increased in plants grown at 15°C and 18°C compared with those grown at 20°C. In all cases GR activity was found only in mesophyll fractions of the leaves, with no GR activity being detectable in bundle sheath extracts. Immunogold labeling with GR-specific antibodies showed that the GR protein was exclusively localized in the mesophyll cells of leaves at all growth temperatures, whereas GR transcripts (as determined by in situ hybridization techniques) were observed in both cell types. These results indicate that post-transcriptional regulation prevents GR accumulation in the bundle sheath cells of maize leaves. The resulting limitation on the capacity for regeneration of reduced glutathione in this compartment may contribute to the extreme chilling sensitivity of maize leaves. PMID:10712529

  19. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Physiology and biochemistry of source-regulated protein accumulation in the wheat grain.

    Science.gov (United States)

    Barneix, Atilio J

    2007-05-01

    Wheat is unique among cereals for the baking qualities of its flour, which are dependent upon the type and concentration of its proteins. As a consequence, the grain protein concentration (GPC) is one of the main determinants of wheat international market price. More than 50-70% of the final grain N is accumulated before flowering and later remobilized to the grain, N fertilization being the common practice used to produce high GPC. However, after incremental additions of N fertilizer, GPC reaches a maximum and then remains constant, without any increase in N uptake or remobilization by the crop, thus decreasing the efficiency of N fertilizer. Although, the genetic and molecular mechanisms that regulate N uptake by the roots are being clarified quickly, the regulation and physiology of N transport from the leaves to the grain remains less clear. In this review, the possible regulatory points involved in N transport to the grain and the difficulties for increasing GPC are discussed. It has been demonstrated that protein synthesis in the grain is source-limited, and that the grain can accumulate protein limited only by the amino acids provided by the phloem. It has also been shown that there is no limitation in the amino acid/sugar ratios that can be exported to the phloem. On the other hand, NO(3)(-) uptake transporters are depressed when the plant concentration of some amino acids, such as glutamine, is high. It has also been shown that a high N supply increases cytokinins concentration, preventing leaf senescence and proteolysis. Based on this information, it is postulated that there are two main regulatory points during grain filling when plant N status is ample. On the one hand, the N uptake transporters in the roots are depressed due to the high amino acids concentration in the tissues, and N uptake is low. On the other, a high amino acids concentration keeps the cytokinins level high, repressing leaf protein degradation and decreasing amino acid export to the

  1. Extractable Bacterial Surface Proteins in Probiotic–Host Interaction

    Directory of Open Access Journals (Sweden)

    Fillipe L. R. do Carmo

    2018-04-01

    Full Text Available Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp, this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs, and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.

  2. 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.

  3. Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p

    International Nuclear Information System (INIS)

    Hagerman, Ruth A.; Waring, Natashya J.; Willis, Richard A.; Hagerman, Ann E.

    2006-01-01

    In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b

  4. Low density lipoprotein receptor-related protein 1 expression correlates with cholesteryl ester accumulation in the myocardium of ischemic cardiomyopathy patients

    Directory of Open Access Journals (Sweden)

    Cal Roi

    2012-08-01

    Full Text Available Abstract Our hypothesis was that overexpression of certain lipoprotein receptors might be related to lipid accumulation in the human ischemic myocardium. Intramyocardial lipid overload contributes to contractile dysfunction and arrhythmias in cardiomyopathy. Thus, the purpose of this study was to assess the effect of hypercholesterolemic LDL and hypertrigliceridemic VLDL dose on LRP1 expression in cardiomyocytes, as well as the potential correlation between LRP1 expression and neutral lipid accumulation in the left ventricle tissue from ischemic cardiomyopathy patients. Cell culture experiments include control and LRP1-deficient cardiomyocytes exposed to lipoproteins under normoxic and hypoxic conditions. Explanted hearts from 18 ICM patients and eight non-diseased hearts (CNT were included. Low density lipoprotein receptor-related protein 1 (LRP1, very low density lipoprotein receptor (VLDLR and low density lipoprotein receptor (LDLR expression was analyzed by real time PCR and Western blotting. Cholesteryl ester (CE, triglyceride (TG and free cholesterol (FC content was assess by thin layer chromatography following lipid extraction. Western blotting experiments showed that protein levels of LRP1, VLDLR and HIF-1α were significantly upregulated in ischemic hearts. Immunohistochemistry and confocal microscopy analysis showed that LRP1 and HIF-1α were upregulated in cardiomyocytes of ICM patients. In vitro studies showed that VLDL, LDL and hypoxia exerted an upregulatory effect on LRP1 expression and that LRP1 played a major role in cholesteryl ester accumulation from lipoproteins in cardiomyocytes. Myocardial CE accumulation strongly correlated with LRP1 levels in ischemic hearts. Taken together, our results suggest that LRP1 upregulation is key for myocardial cholesterol ester accumulation in ischemic human hearts and that LRP1 may be a target to prevent the deleterious effects of myocardial cholesterol accumulation in ischemic cardiomyopathy.

  5. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  6. 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.

  7. 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...

  8. Relationship of non-protein thiol pools and accumulated Cd or Hg in the marine macrophyte Posidonia oceanica (L.) Delile

    International Nuclear Information System (INIS)

    Maserti, B.E.; Ferrillo, V.; Avdis, O.; Nesti, U.; Di Garbo, A.; Catsiki, A.; Maestrini, P.L.

    2005-01-01

    The accumulation of cadmium or mercury and the effect of these elements on the levels of non-protein thiols in the blades of the marine macrophyte Posidonia oceanica were investigated. A significant accumulation of cadmium or mercury, dependent on metal concentration supplied, was observed in metal-treated blades. In the blades treated either with cadmium or mercury, a significant increase in the levels of non-protein thiols (other than glutathione) and a marked depletion of the reduced glutathione content as a function of the metal, exposure time and metal concentration supplied were found. This investigation provides first experimental report on the relationship between non-protein thiol pools and accumulated cadmium or mercury in P. oceanica

  9. 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.

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

    Science.gov (United States)

    Yang, Tsai-Hsiu; Lai, Ying-Hsiu; Lin, Tsuey-Pin; Liu, Wen-Sheng; Kuan, Li-Chun; Liu, Chia-Chyuan

    2014-01-01

    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. PMID:24463291

  11. 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.

  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. 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.

  14. Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[OPEN

    Science.gov (United States)

    2015-01-01

    The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets. PMID:26362606

  15. The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

    Science.gov (United States)

    Li, Shao-jia; Yin, Xue-ren; Xie, Xiu-lan; Allan, Andrew C; Ge, Hang; Shen, Shu-ling; Chen, Kun-song

    2016-02-03

    Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

  16. 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.

  17. p53-inducible DHRS3 Is an Endoplasmic Reticulum Protein Associated with Lipid Droplet Accumulation*

    Science.gov (United States)

    Deisenroth, Chad; Itahana, Yoko; Tollini, Laura; Jin, Aiwen; Zhang, Yanping

    2011-01-01

    The transcription factor p53 plays a critical role in maintaining homeostasis as it relates to cellular growth, proliferation, and metabolism. In an effort to identify novel p53 target genes, a microarray approach was utilized to identify DHRS3 (also known as retSDR1) as a robust candidate gene. DHRS3 is a highly conserved member of the short chain alcohol dehydrogenase/reductase superfamily with a reported role in lipid and retinoid metabolism. Here, we demonstrate that DHRS3 is an endoplasmic reticulum (ER) protein that is shuttled to the ER via an N-terminal endoplasmic reticulum targeting signal. One important function of the ER is synthesis of neutral lipids that are packaged into lipid droplets whose biogenesis occurs from ER-derived membranes. DHRS3 is enriched at focal points of lipid droplet budding where it also localizes to the phospholipid monolayer of ER-derived lipid droplets. p53 promotes lipid droplet accumulation in a manner consistent with DHRS3 enrichment in the ER. As a p53 target gene, the observations of Dhrs3 location and potential function provide novel insight into an unexpected role for p53 in lipid droplet dynamics with implications in cancer cell metabolism and obesity. PMID:21659514

  18. 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

  19. 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

  20. 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.

  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. 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.

  3. Net herbage accumulation rate and crude protein content of Urochloa brizantha cultivars under shade intensities

    Directory of Open Access Journals (Sweden)

    Paulo Roberto de Lima Meirelles

    2013-12-01

    Full Text Available The use of silvopastoral systems is a sustainable alternative for animal production in various regions of the Brazil. However to obtain satisfactory results in these systems, the selection of forage species that grows well in the shade should be done. The tolerance of plants to light restriction and the correctly choice of species, considering good nutritional values for these conditions has great importance. The study of artificial shading for forage production helps the clarification of issues related to the behavior of plants under reduced light prior to use in integrations with forests. The aim of the study was to evaluate the net herbage accumulation rate of forage (HAR and crude protein (CP of Urochloa brizantha cultivars (Marandu and Piatã under natural light and shading of 30 and 60%. The experiment was conducted at FMVZ - UNESP, Botucatu. The experimental design was a randomized block in factorial arrangement 3 x 2 (three shading levels: 0, 30 and 60%, two cultivars: Marandu and Piatã with three replications and repeated measures (3 cuts. Sample collection occurred when the cultivars reached 35 cm in height. The treatments with shading showed lower cutting intervals as compared to those subjected to full sunlight, because they have reached in a shorter time to time as determined cut-off criterion (mean of 37, 45 and 61 days for reduction of 60%, reduction of 30% and full sun. Significant effects (P<0.05 interaction cultivar x shade x cut on the net herbage accumulation rate (HAR. Most HAR (P<0.05 was observed for cv. Marandu 60% reduction in lightness (127 kg/ha/day due to increased production of stem during the first growing cycle. The lower HAR also occurred to Marandu, but under natural light in the third cut (34 kg/ha/day due to adverse weather conditions during the growth interval. The shadow effect and the cutting (P<0.05 affected CP. The percentage of CP on cultivars showed the highest values (average value of 9.27% in 60

  4. The fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex.

    Science.gov (United States)

    Garcia-Higuera, I; Kuang, Y; Denham, J; D'Andrea, A D

    2000-11-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The FANCG protein binds directly to the amino terminal nuclear localization sequence (NLS) of FANCA, suggesting that FANCG plays a role in regulating FANCA nuclear accumulation. In the current study the functional consequences of FANCG/FANCA binding were examined. Correction of an FA-G cell line with the FANCG complementary DNA (cDNA) resulted in FANCA/FANCG binding, prolongation of the cellular half-life of FANCA, and an increase in the nuclear accumulation of the FA protein complex. Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG. Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm. The mutant forms failed to translocate to the nucleus of transduced cells, thereby suggesting a model of coordinated binding and nuclear translocation. These results demonstrate that the FANCA/FANCG interaction is required to maintain the cellular levels of both proteins. Moreover, at least one function of FANCG and FANCA is to regulate the nuclear accumulation of the FA protein complex. Failure to accumulate the nuclear FA protein complex results in the characteristic spectrum of clinical and cellular abnormalities observed in FA.

  5. An effective protein extraction method for two-dimensional electrophoresis in the anticancer herb Andrographis paniculata Nees.

    Science.gov (United States)

    Talei, Daryush; Valdiani, Alireza; Puad, Mohd Abdullah

    2013-01-01

    Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances, including polysaccharides, polyphenols, and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein content among the four methods. The chloroform-trichloroacetic acid-acetone method using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer provided the best results in terms of protein content, pellets, spot resolution, and intensity of unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2-DE gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies of Andrographis paniculata, which may also be applied to other recalcitrant medicinal plant tissues. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  6. Efficient extraction of protein-protein interactions from full-text articles.

    Science.gov (United States)

    Hakenberg, Jörg; Leaman, Robert; Vo, Nguyen Ha; Jonnalagadda, Siddhartha; Sullivan, Ryan; Miller, Christopher; Tari, Luis; Baral, Chitta; Gonzalez, Graciela

    2010-01-01

    Proteins and their interactions govern virtually all cellular processes, such as regulation, signaling, metabolism, and structure. Most experimental findings pertaining to such interactions are discussed in research papers, which, in turn, get curated by protein interaction databases. Authors, editors, and publishers benefit from efforts to alleviate the tasks of searching for relevant papers, evidence for physical interactions, and proper identifiers for each protein involved. The BioCreative II.5 community challenge addressed these tasks in a competition-style assessment to evaluate and compare different methodologies, to make aware of the increasing accuracy of automated methods, and to guide future implementations. In this paper, we present our approaches for protein-named entity recognition, including normalization, and for extraction of protein-protein interactions from full text. Our overall goal is to identify efficient individual components, and we compare various compositions to handle a single full-text article in between 10 seconds and 2 minutes. We propose strategies to transfer document-level annotations to the sentence-level, which allows for the creation of a more fine-grained training corpus; we use this corpus to automatically derive around 5,000 patterns. We rank sentences by relevance to the task of finding novel interactions with physical evidence, using a sentence classifier built from this training corpus. Heuristics for paraphrasing sentences help to further remove unnecessary information that might interfere with patterns, such as additional adjectives, clauses, or bracketed expressions. In BioCreative II.5, we achieved an f-score of 22 percent for finding protein interactions, and 43 percent for mapping proteins to UniProt IDs; disregarding species, f-scores are 30 percent and 55 percent, respectively. On average, our best-performing setup required around 2 minutes per full text. All data and pattern sets as well as Java classes that

  7. The duck hepatitis B virus polymerase and core proteins accumulate in different patterns from their common mRNA

    International Nuclear Information System (INIS)

    Yao Ermei; Schaller, Heinz; Tavis, John E.

    2003-01-01

    Hepadnaviral reverse transcription occurs in capsids in which the core (C) protein surrounds the reverse transcriptase (P) and pregenomic RNA (pgRNA). We analyzed the accumulation patterns of duck hepatitis B virus P, C, and pgRNA in transfected LMH cells, infected primary duck hepatocytes (PDH), and infected duck liver. In all three systems, P accumulated over time in a different pattern compared with C, despite translation of both proteins from the pgRNA. Although the accumulation patterns of the proteins varied between the systems, in each case P became detectable at the same time or earlier than C and the ratio of P relative to C dropped with time. These accumulation patterns were consistent with the translation rates and half-lives of P and C. Comparing the translation rates of P and C with the pgRNA level over time revealed that translation of P and C was negatively regulated in LMH cells. These data provide a framework for comparing replication studies performed in LMH cells, PDHs and ducks

  8. 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.

  9. 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.

  10. 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

  11. Tula hantavirus NSs protein accumulates in the perinuclear area in infected and transfected cells.

    Science.gov (United States)

    Virtanen, Jussi Oskari; Jääskeläinen, Kirsi Maria; Djupsjöbacka, Janica; Vaheri, Antti; Plyusnin, Alexander

    2010-01-01

    The small RNA segment of some hantaviruses (family Bunyaviridae) encodes two proteins: the nucleocapsid protein and, in an overlapping reading frame, a non-structural (NSs) protein. The hantavirus NSs protein, like those of orthobunya- and phleboviruses, counteracts host innate immunity. Here, for the first time, the NSs protein of a hantavirus (Tula virus) has been observed in infected cells and shown to localize in the perinuclear area. Transiently expressed NSs protein showed similar localization, although the kinetics was slightly different, suggesting that to reach its proper location in the infected cell, the NSs protein does not have to cooperate with other viral proteins.

  12. The peculiarities of the accumulation of Cs-137 and the content of photosynthetic pigments and protein in plants of natural associations of south-east Belarus

    International Nuclear Information System (INIS)

    Gaponenko, V. I.; Kravchenko, V. A; Matsko, V. P.; Grushevskaya, O. M.

    1994-01-01

    The peculiarities of accumulation of Cs-137 by various components of meadow associations of Polesky State radioecological reserve (PSRER) were studied. The specific radioactivity of soil totalled 12000-290000 Bq/kg, and specific radioactivity of overground phytomass was 200-252000 Bq/kg. The inverse dependence between the specific radioactivity and the coefficient of accumulation of plants was observed, and the direct dependence between the acidity of salt extract (1H KCL) and the coefficient of accumulation. The content of photosynthetic pigments of Agropyron repens L. was less in the phase of florescence and fruitage than at the beginning of vegetation. That is said about the principal decrease of the content of chlorophyll b comparing with chlorophyll a and the both green pigments comparing with carotenoids. The fourth month's period of vegetation of plants in the conditions of increased radiation background was not observed the changing neither by the of chlorophyll a and b and carotinoids nor by the content of total protein that was testified about the high stability of pigmental system. (author)

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

    International Nuclear Information System (INIS)

    Song, Jun; Ren, Pingping; Zhang, Lin; Wang, Xing Li; Chen, Li; Shen, Ying H.

    2010-01-01

    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.

  14. 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.

  15. Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco

    International Nuclear Information System (INIS)

    Yalpani, N.; Enyedi, A.J.; León, J.; Raskin, I.

    1994-01-01

    In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 μg·g −1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance. (author)

  16. Differentially Accumulated Proteins in Coffea arabica Seeds during Perisperm Tissue Development and Their Relationship to Coffee Grain Size.

    Science.gov (United States)

    Alves, Leonardo Cardoso; Magalhães, Diogo Maciel De; Labate, Mônica Teresa Veneziano; Guidetti-Gonzalez, Simone; Labate, Carlos Alberto; Domingues, Douglas Silva; Sera, Tumoru; Vieira, Luiz Gonzaga Esteves; Pereira, Luiz Filipe Protasio

    2016-02-24

    Coffee is one of the most important crops for developing countries. Coffee classification for trading is related to several factors, including grain size. Larger grains have higher market value then smaller ones. Coffee grain size is determined by the development of the perisperm, a transient tissue with a highly active metabolism, which is replaced by the endosperm during seed development. In this study, a proteomics approach was used to identify differentially accumulated proteins during perisperm development in two genotypes with regular (IPR59) and large grain sizes (IPR59-Graudo) in three developmental stages. Twenty-four spots were identified by MALDI-TOF/TOF-MS, corresponding to 15 proteins. We grouped them into categories as follows: storage (11S), methionine metabolism, cell division and elongation, metabolic processes (mainly redox), and energy. Our data enabled us to show that perisperm metabolism in IPR59 occurs at a higher rate than in IPR59-Graudo, which is supported by the accumulation of energy and detoxification-related proteins. We hypothesized that grain and fruit size divergences between the two coffee genotypes may be due to the comparatively earlier triggering of seed development processes in IPR59. We also demonstrated for the first time that the 11S protein is accumulated in the coffee perisperm.

  17. Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis--an approach for slowing Alzheimer disease?

    Science.gov (United States)

    Bendiske, Jennifer; Bahr, Ben A

    2003-05-01

    Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.

  18. Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

    OpenAIRE

    Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

    2011-01-01

    Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit t...

  19. Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

    Science.gov (United States)

    Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

    2008-02-22

    One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

  20. A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation.

    Science.gov (United States)

    Kupfer, G; Naf, D; Garcia-Higuera, I; Wasik, J; Cheng, A; Yamashita, T; Tipping, A; Morgan, N; Mathew, C G; D'Andrea, A D

    1999-04-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but the function of the encoded FA proteins remains unknown. We recently demonstrated that the FANCA and FANCC proteins bind and form a nuclear complex. In the current study, we identified a homozygous mutation in the FANCA gene (3329A>C) in an Egyptian FA patient from a consanguineous family. This mutant FANCA allele is predicted to encode a mutant FANCA protein, FANCA(H1110P), in which histidine 1110 is changed to proline. Initially, we characterized the FANCA(H1110P) protein, expressed in an Epstein Barr virus (EBV)-immortalized lymphoblast line derived from the patient. Unlike wild-type FANCA protein expressed in normal lymphoblasts, FANCA(H1110P) was not phosphorylated and failed to bind to FANCC. To test directly the effect of this mutation on FANCA function, we used retroviral-mediated transduction to express either wild-type FANCA or FANCA(H1110P) protein in the FA-A fibroblast line, GM6914. Unlike wild-type FANCA, the mutant protein failed to complement the mitomycin C sensitivity of these cells. In addition, the FANCA(H1110P) protein was defective in nuclear accumulation in the transduced cells. The characteristics of this mutant protein underscore the importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation in the function of the FA pathway.

  1. Calculating the Degradation Rate of Individual Proteins Using Xenopus Extract Systems.

    Science.gov (United States)

    McDowell, Gary S; Philpott, Anna

    2018-05-16

    The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts. © 2019 Cold Spring Harbor Laboratory Press.

  2. 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

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

    Science.gov (United States)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

    2015-12-01

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa 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-∆RTCter) 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-∆RTCter. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. 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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Zanthoxylum piperitum DC ethanol extract suppresses fat accumulation in adipocytes and high fat diet-induced obese mice by regulating adipogenesis.

    Science.gov (United States)

    Gwon, So Young; Ahn, Ji Yun; Kim, Tae Wan; Ha, Tae Youl

    2012-01-01

    This study was conducted to determine the anti-obesity effects of Zanthoxylum piperitum DC fruit ethanol extract (ZPE) in 3T3-L1 adipocytes and obese mice fed a high-fat diet. We evaluated the influence of the addition of ZPE to a high-fat diet on body weight, adipose tissue weight, serum and hepatic lipids in C57BL/6 mice. In addition, adipogenic gene expression was determined by Western blot and real-time reverse transcription-PCR analysis. We assessed the effect of ZPE on 3T3-L1 preadipocyte differentiation. ZPE reduced weight gain, white adipose tissue mass, and serum triglyceride and cholesterol levels (pZPE decreased lipid accumulation and PPARγ, C/EBPα, SREBP-1, and FAS protein and mRNA levels in the liver. ZPE inhibited in vitro adipocyte differentiation in a dose-dependent manner and significantly attenuated adipogenic transcription factors, such as PPARγ, C/EBPα, and SREBP-1 in 3T3L1 cells. These findings suggest that Z. piperitum DC exerts an anti-obesity effect by inhibiting adipogenesis through the downregulation of genes involved in the adipogenesis pathway.

  6. 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

  7. 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.

  8. The use of solid sorbents for direct accumulation of organic compounds from water matrices : a review of solid-phase extraction techniques

    NARCIS (Netherlands)

    Liska, I.; Krupcik, J.; Leclercq, P.A.

    1989-01-01

    The main principles of solid-phase extraction techniques are reviewed in this paper. Various solid sorbents can be used as a suitable trap for direct accumulation of organic compounds from aqueous solutions. The trapped analytes can be desorbed by elution with suitably chosen liquid phases. These

  9. Extracts of black and brown rice powders improve hepatic lipid accumulation via the activation of PPARα in obese and diabetic model mice.

    Science.gov (United States)

    Felix, Angelina Dr; Takahashi, Nobuyuki; Takahashi, Mami; Katsumata-Tsuboi, Rie; Satoh, Ryo; Soon Hui, Teoh; Miyajima, Katsuhiro; Nakae, Dai; Inoue, Hirofumi; Uehara, Mariko

    2017-11-01

    Rice powder extract (RPE) from black and brown rice (Oryza sativa L. indica) improves hepatic lipid accumulation in obese and diabetic model mice via peroxisomal fatty acid oxidation. RPE showed PPARα agonistic activity which did not differ between black and brown RPE despite a higher anthocyanin content in black RPE.

  10. Supplementation with Vitis vinifera L. skin extract improves insulin resistance and prevents hepatic lipid accumulation and steatosis in high-fat diet-fed mice.

    Science.gov (United States)

    Santos, Izabelle Barcellos; de Bem, Graziele Freitas; Cordeiro, Viviane Silva Cristino; da Costa, Cristiane Aguiar; de Carvalho, Lenize Costa Reis Marins; da Rocha, Ana Paula Machado; da Costa, Gisele França; Ognibene, Dayane Teixeira; de Moura, Roberto Soares; Resende, Angela Castro

    2017-07-01

    Nonalcoholic fatty liver disease is one of the most common complications of obesity. The Vitis vinifera L. grape skin extract (ACH09) is an important source of polyphenols, which are related to its antioxidant and antihyperglycemic activities. We hypothesized that ACH09 could also exert beneficial effects on metabolic disorders associated with obesity and evaluated ACH09's influence on high-fat (HF) diet-induced hepatic steatosis and insulin resistance in C57BL/6 mice. The animals were fed a standard diet (10% fat, control) or an HF diet (60% fat, HF) with or without ACH09 (200mg/[kg d]) for 12weeks. Our results showed that ACH09 reduced HF diet-induced body weight gain, prevented hepatic lipid accumulation and steatosis, and improved hyperglycemia and insulin resistance. The underlying mechanisms of these beneficial effects of ACH09 may involve the activation of hepatic insulin-signaling pathway because the expression of phosphorylated insulin receptor substrate-1, phosphatidylinositol 3-kinase, phosphorylated Akt serine/threonine kinase 1, and glucose transporter 2 was increased by ACH09 and correlated with improvement of hyperglycemia, hyperinsulinemia, and insulin resistance. ACH09 reduced the expression of the lipogenic factor sterol regulatory-element binding protein-1c in the liver and upregulated the lipolytic pathway (phosphorylated liver kinase B1/phosphorylated adenosine-monophosphate-activated protein kinase), which was associated with normal hepatic levels of triglyceride and cholesterol and prevention of steatosis. ACH09 prevented the hepatic oxidative damage in HF diet-fed mice probably by restoration of antioxidant activity. In conclusion, ACH09 protected mice from HF diet-induced obesity, insulin resistance, and hepatic steatosis. The regulation of hepatic insulin signaling pathway, lipogenesis, and oxidative stress may contribute to ACH09's protective effect. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. 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.

  12. Proteomic Analysis of Differentially Accumulated Proteins in Cucumber (Cucumis sativus) Fruit Peel in Response to Pre-storage Cold Acclimation.

    Science.gov (United States)

    Wang, Bin; Shen, Fei; Zhu, Shijiang

    2017-01-01

    Harvested fruits are still living organs and respond to environmental stimuli. Low temperature storage is effective in extending life of harvested fruit, but it may also cause chilling injury. Cold acclimation has been shown to induce chilling tolerance in plants, but what proteomic changes caused by cold acclimation are related to defense against chilling stress remains largely unclear. Here, 3 d of pre-storage cold acclimation (PsCA) at 10°C reduced chilling injury and secondary disease severity in cucumber stored at 5°C by 51 and 94%, respectively, compared with the control which was directly stored at 5°C. Proteomic analysis of cucumber peel identified 21 significant differentially-accumulated proteins (SDAPs) right after PsCA treatment and 23 after the following cold storage (PsCA+CS). These proteins are mainly related to stress response and defense (SRD), energy metabolism, protein metabolism, signal transduction, primary metabolism, and transcription. The SRD proteins, which made up 37% of the 21 and 47% of the 23, respectively, represented the largest class of SDAPs, and all but one protein were up-regulated, suggesting accumulation of proteins involved in defense response is central feature of proteomic profile changes brought about by PsCA. In fruit just after PsCA treatment, the identified SDAPs are related to responses to various stresses, including chilling, salt stress, dehydration, fungi, bacteria, insects, and DNA damage. However, after prolonged cold storage, the targeted proteins in acclimated fruit were narrowed down in scope to those involved in defense against chilling and pathogens. The change patterns at the transcription level of the majority of the up-regulated differentially-accumulated proteins were highly consistent with those at protein level. Taken all, the results suggest that the short-time cold acclimation initiated comprehensive defense responses in cucumber fruit at first, while the long term storage thereafter altered the

  13. Proteomic Analysis of Differentially Accumulated Proteins in Cucumber (Cucumis sativus Fruit Peel in Response to Pre-storage Cold Acclimation

    Directory of Open Access Journals (Sweden)

    Bin Wang

    2018-01-01

    Full Text Available Harvested fruits are still living organs and respond to environmental stimuli. Low temperature storage is effective in extending life of harvested fruit, but it may also cause chilling injury. Cold acclimation has been shown to induce chilling tolerance in plants, but what proteomic changes caused by cold acclimation are related to defense against chilling stress remains largely unclear. Here, 3 d of pre-storage cold acclimation (PsCA at 10°C reduced chilling injury and secondary disease severity in cucumber stored at 5°C by 51 and 94%, respectively, compared with the control which was directly stored at 5°C. Proteomic analysis of cucumber peel identified 21 significant differentially-accumulated proteins (SDAPs right after PsCA treatment and 23 after the following cold storage (PsCA+CS. These proteins are mainly related to stress response and defense (SRD, energy metabolism, protein metabolism, signal transduction, primary metabolism, and transcription. The SRD proteins, which made up 37% of the 21 and 47% of the 23, respectively, represented the largest class of SDAPs, and all but one protein were up-regulated, suggesting accumulation of proteins involved in defense response is central feature of proteomic profile changes brought about by PsCA. In fruit just after PsCA treatment, the identified SDAPs are related to responses to various stresses, including chilling, salt stress, dehydration, fungi, bacteria, insects, and DNA damage. However, after prolonged cold storage, the targeted proteins in acclimated fruit were narrowed down in scope to those involved in defense against chilling and pathogens. The change patterns at the transcription level of the majority of the up-regulated differentially-accumulated proteins were highly consistent with those at protein level. Taken all, the results suggest that the short-time cold acclimation initiated comprehensive defense responses in cucumber fruit at first, while the long term storage thereafter

  14. Multiple layers of temporal and spatial control regulate accumulation of the fruiting body-specific protein APP in Sordaria macrospora and Neurospora crassa.

    Science.gov (United States)

    Nowrousian, Minou; Piotrowski, Markus; Kück, Ulrich

    2007-07-01

    During fungal fruiting body development, specialized cell types differentiate from vegetative mycelium. We have isolated a protein from the ascomycete Sordaria macrospora that is not present during vegetative growth but accumulates in perithecia. The protein was sequenced by mass spectrometry and the corresponding gene was termed app (abundant perithecial protein). app transcript occurs only after the onset of sexual development; however, the formation of ascospores is not a prerequisite for APP accumulation. The transcript of the Neurospora crassa ortholog is present prior to fertilization, but the protein accumulates only after fertilization. In crosses of N. crassa Deltaapp strains with the wild type, APP accumulates when the wild type serves as female parent, but not in the reciprocal cross; thus, the presence of a functional female app allele is necessary and sufficient for APP accumulation. These findings highlight multiple layers of temporal and spatial control of gene expression during fungal development.

  15. HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPARγ

    International Nuclear Information System (INIS)

    Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin; Park, Min Jung; Kim, Kwang Jin; Cheong, JaeHun

    2007-01-01

    Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPARγ (peroxisome proliferators-activated receptor γ) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPARγ. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfected with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPARγ transcriptional activity. However, HCV core protein had no effect on PPARγ gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection

  16. MIPCE: An MI-based protein complex extraction technique

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... In this work, a method is proposed, referred to as. MIPCE, to find protein ... Because of its importance in the studies of protein interaction network, there are ..... three publicly available benchmark real-life datasets. The method was ... protein interaction networks with improved balance and scal- ability; in ...

  17. 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.

  18. Effects of Chowiseungcheng-tang Extracts on the Preadipocytes Proliferation in 3T3-L1 cell line, Lipolysis of Adipocytes in rat, and Localized Fat Accumulation by extraction methods

    Directory of Open Access Journals (Sweden)

    Jae-eun, Lee

    2008-03-01

    Full Text Available Objectives : The purpose of this study is to investigate the effects of Chowiseungcheng-tang extracts on the preadipocytes proliferation in 3T3-L1 cell line, lipolysis of adipocytes in rat’s epididymal adipocytes and localized fat accumulation of porcine by extraction methods(alcohol and water. Methods : Diminish preadipocytes proliferation and promote lipolysis of adipocytes do primary role to reduce obesity. So, we used 3T3-L1 mouse embryo fibroblasts(preadipocytes and rat epididymal adipocytes from Sprague-Dawley rats to investigate the effects of Chowiseungcheng-tang extracts on the preadipocytes proliferation, lipolysis of adipocytes. They were treated with 0.01, 0.1, 1.0㎎/㎖ Chowiseungcheng-tang alcohol and water extracts. And for the purpose of investigating the effects of Chowiseungcheng-tang alcohol and water extracts on the localized fat accumulation, we injected 0.1, 1.0, 10.0㎎/㎖ Chowiseungcheng-tang extracts to porcine fat tissues and observed histological changes of them. Results : Following results were obtained from the preadipocytes proliferation and lipolysis of adipocytes and histological investigation of fat tissues. 1. Chowiseungcheng-tang extracts suppressed preadipocytes proliferation on the high dosage(especially 1.0㎎/㎖, and especially alcohol extracts had better effects. 2. The alcohol extracts of Chowiseungcheng-tang decreased the activity of glycerol-3-phosphate dehydrogenase(GPDH on the concentrations of 0.1, 1.0㎎/㎖. Alcohol extracts had better effects than water extracts. 3. Chowiseungcheng-tang extracts increased lipolysis of adipocytes on the concentrations of 0.1, 1.0㎎/㎖, and especially on the concentration of 1.0㎎/㎖ alcohol extract of Chowiseungcheng-tang had better effect. 4. The water extract of Chowiseungcheng-tang had significant activity to the destruction of porcine fat cell membranes only on the concentration of 10.0㎎/㎖, but alcohol extracts of Chowiseungcheng-tang had it on all

  19. Effect of compounds astragalus extract and cysteamine on finishing pigs growth and protein turnover

    International Nuclear Information System (INIS)

    Wang Cheng; Wang Zhisheng; Zhou Anguo; Liu Guilian; Xue Bai

    2008-01-01

    32 PIC pigs, weighted 59.82±1.32 kg on average, were subjected randomly to 4 treatments for determining the effects of extrogenous metabolism-regulating agents on pig performance, protein turn-over and nitrogen retention by using isotope ( 15 N-Gly) technique and nitrogen balance trial. The 4 treatments were: (1) basal diet; (2) basal diet plus cysteamine (70mg/kg, CS group); (3) basal diet plus non-stable astragalus extracts 250mg/kg (NCAE group); (4) basal diet plus stably astragalus extracts 250mg/kg (CAE group). Astragalus extracts and cysteamine, especially NCAE, redounded to increase pig performance, especially increase average day gain (P<0.05). Both astragalus extracts and cysteamine accelerated protein aggradation rate and amino acid utilization rate, as well as biological value. Astragalus extracts helped to accelerate the synthesis rate of amino acid into body protein and reduce the excretion rate of endogenous urine nitrogen. The mechanism of increasing protein deposition of cysteamine is reducing body protein degradation rate (26.71% reduction, compared to control group), while that of astragalus extracts is bilateral, reducing protein degradation rate (24.84% and 3.66% reduction for NCAE and CAE) and accelerating protein synthesis rate (22.86% and 19.18% increase for NCAE and CAE). Both astragalus extracts and cysteamine contributed the increasing nitrogen retention, net nitrogen utilization and biological value. (authors)

  20. Atorvastatin reduces lipid accumulation in the liver by activating protein kinase A-mediated phosphorylation of perilipin 5.

    Science.gov (United States)

    Gao, Xing; Nan, Yang; Zhao, Yuanlin; Yuan, Yuan; Ren, Bincheng; Sun, Chao; Cao, Kaiyu; Yu, Ming; Feng, Xuyang; Ye, Jing

    2017-12-01

    Statins have been proven to be effective in treating non-alcoholic fatty liver disease (NAFLD). Recently, it was reported that statins decreased the hepatic expression of perilipin 5 (Plin5), a lipid droplet (LD)-associated protein, which plays critical roles in regulating lipid accumulation and lipolysis in liver. However, the function and regulation mechanism of Plin5 have not yet been well-established in NAFLD treatment with statins. In this study, we observed that atorvastatin moderately reduced the expression of Plin5 in livers without changing the protein level of Plin5 in the hepatic LD fraction of mice fed with high-fat diet (HFD). Intriguingly, atorvastatin stimulated the PKA-mediated phosphorylation of Plin5 and reduced the triglyceride (TG) accumulation in hepatocytes with overexpression of wide type (Plin5-WT) compared to serine-155 mutant Plin5 (Plin5-S155A). Moreover, PKA-stimulated FA release of purified LDs carrying Plin5-WT but not Plin5-S155A. Glucagon, a PKA activator, stimulated the phosphorylation of Plin5-WT and inhibited its interaction with CGI-58. The results indicated that atorvastatin promoted lipolysis and reduced TG accumulation in the liver by increasing PKA-mediated phosphorylation of Plin5. This new mechanism of lipid-lowering effects of atorvastatin might provide a new strategy for NAFLD treatment. Copyright © 2017. Published by Elsevier B.V.

  1. 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.

  2. Extractable proteins from electron beam (EB) irradiated natural rubber (NR) latex

    International Nuclear Information System (INIS)

    Feroza Akhtar; Fumio Yoshii; Keizo Makuuchi

    1996-01-01

    The protein assay of natural rubber latex (NRL) vulcanized by low energy electron beam (EB, 300 keV, 30 mA) has been carried out using Bicinchoninic acid (BCA) reagent. Extractable protein in irradiated latex film was determined by measuring the absorption of colored solution at 562 nm using UV spectrometer. The effect of various radiation doses on the extractable protein content of NRL was investigated. It was ,found that the quantities of extractable protein increases with radiation dose. When compared with ,gamma-ray irradiated samples the same trend was observed. Electron beam irradiated latex films are leached in 1% (ammonia water for various lengths of time. From the results it was established that within 2 hours of leaching in ammonia water most of the extractable protein (96%) were removed from rubber film

  3. Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

    International Nuclear Information System (INIS)

    Banzet, N.; Richaud, C.; Deveaux, Y.; Kazmaier, M.; Gagnon, J.; Triantaphylides, C.

    1998-01-01

    Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential

  4. 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.

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

    International Nuclear Information System (INIS)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

    2015-01-01

    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_C_t_e_r) 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_C_t_e_r. 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_C_t_e_r 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.

  6. Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

    Science.gov (United States)

    Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

    2011-01-01

    Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets. PMID:21857022

  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

    We previously found that dietary soy protein produces higher lipoprotein(a) [Lp(a)] plasma concentrations than does casein. This study tested the hypothesis that soy protein contains Lp(a)-raising alcohol-removable components. Twelve normolipidemic women and men consumed, in a crossover design......, 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 (Psoy 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...

  8. 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.

  9. Green tea extract impairs meat emulsion properties by disturbing protein disulfide cross-linking.

    Science.gov (United States)

    Jongberg, Sisse; Terkelsen, Linda de S; Miklos, Rikke; Lund, Marianne N

    2015-02-01

    The dose-dependent effects of green tea extract (100, 500, or 1500ppm) on the textural and oxidative stability of meat emulsions were investigated, and compared to a control meat emulsion without extract. All levels of green tea extract inhibited formation of TBARS as a measure for lipid oxidation. Overall protein thiol oxidation and myosin heavy chain (MHC) cross-linking were inhibited by 100ppm green tea extract without jeopardizing the textural stability, while increasing concentrations of extract resulted in reduced thiol concentration and elevated levels of non-reducible protein modifications. Addition of 1500ppm green tea extract was found to modify MHC as evaluated by SDS-PAGE combining both protein staining and specific thiol staining, indicating that protein modifications generated through reactions of green tea phenolic compounds with protein thiols, disrupted the meat emulsion properties leading to reduced water holding capacity and textural stability. Hence, a low dose of green tea extract preserves both the textural and the oxidative stability of the meat proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Xu, Kaijia; Wang, Yuzhi; Huang, Yanhua; Li, Na; Wen, Qian

    2015-01-01

    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

  11. 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

  12. 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

  13. 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.

  14. 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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. 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.

  16. 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

  17. Protein extraction from the stem of Panax ginseng C. A. Meyer: A ...

    African Journals Online (AJOL)

    Administrator

    2011-05-23

    May 23, 2011 ... Meyer: A tissue of lower protein extraction efficiency for proteomic analysis ..... key technology R&D program (No. 2007BAI38B02),. National ... Six new dammarane-type triterpene saponins from the leaves of. Panax ginseng.

  18. 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.

  19. 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

    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......-insensitive GS knock-in mice, although AICAR-stimulated AMPK activation, glucose transport, and total glucose utilization were normal. CONCLUSIONS We provide genetic evidence that AMPK activation promotes muscle glycogen accumulation by allosteric activation of GS through an increase in glucose uptake...

  20. A comparison of tissue preparation methods for protein extraction of cocoa (Theobroma cacao L. pod

    Directory of Open Access Journals (Sweden)

    Ascensión Martínez-Márquez

    2017-04-01

    Full Text Available Cocoa, Theobroma cacao L. is one of the main tropical industrial crops. Cocoa has a very high level of interfering substances, such as polysaccharides and phenolic compounds that could prevent the isolation of suitable protein. Efficient methods of protein extraction are a priority to successfully apply proteomic analyses. We compared and evaluated two methods (A and B of tissue preparation for total protein extract by phenol/SDS extraction protocol. The difference in the application of the two methods was that extensively washed dry powder of pod tissue were made in Method A, whereas that crude extract were prepared Method B. Extracted proteins were examined using one-dimensional electrophoresis (1-D. Results show that each extraction method isolated a unique subset of cocoa pod proteome. Principal component analysis showed little variation in the data obtained using Method A, while that in Methods B showed no low reproducibility, thus demonstrating that Method A is a reliable for preparing cocoa pod proteins. The protocol is expected to be applicable to other recalcitrant plant tissues and to be of interest to laboratories involved in plant proteomics analyses. A combination of extraction approaches is recommended for increasing proteome coverage when using gel-based isolation techniques.

  1. 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

  2. A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

    Science.gov (United States)

    Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

    2017-11-01

    Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  3. 5-Lipoxygenase-Activating Protein as a Modulator of Olanzapine-Induced Lipid Accumulation in Adipocyte

    Directory of Open Access Journals (Sweden)

    Svetlana Dzitoyeva

    2013-01-01

    Full Text Available Experiments were performed in 3T3-L1 preadipocytes differentiated in vitro into adipocytes. Cells were treated with olanzapine and a 5-lipoxygenase (5-LOX activating protein (FLAP inhibitor MK-886. Lipid content was measured using an Oil Red O assay; 5-LOX and FLAP mRNA content was measured using quantitative real-time PCR; the corresponding protein contents were measured using quantitative Western blot assay. Olanzapine did not affect the cell content of 5-LOX mRNA and protein; it decreased FLAP mRNA and protein content at day five but not 24 hours after olanzapine addition. In the absence of MK-886, low concentrations of olanzapine increased lipid content only slightly, whereas a 56% increase was induced by 50 μM olanzapine. A 5-day cotreatment with 10 μM MK-886 potentiated the lipid increasing action of low concentrations of olanzapine. In contrast, in the presence of 50 μM olanzapine nanomolar and low micromolar concentrations of MK-886 reduced lipid content. These data suggest that FLAP system in adipocytes is affected by olanzapine and that it may modify how these cells respond to the second-generation antipsychotic drugs (SGADs. Clinical studies could evaluate whether the FLAP/5-LOX system could play a role in setting a variable individual susceptibility to the metabolic side effects of SGADs.

  4. Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas

    International Nuclear Information System (INIS)

    Warchalowska-Sliwa, E.; Niklinska, M.; Goerlich, A.; Michailova, P.; Pyza, E.

    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 2 and M 3 bivalents in addition to the presence of B chromosomes. - Metals accumulation caused genotoxicity in insects

  5. Extraction of intracellular protein from Chlorella pyrenoidosa using a combination of ethanol soaking, enzyme digest, ultrasonication and homogenization techniques.

    Science.gov (United States)

    Zhang, Ruilin; Chen, Jian; Zhang, Xuewu

    2018-01-01

    Due to the rigid cell wall of Chlorella species, it is still challenging to effectively extract significant amounts of protein. Mass methods were used for the extraction of intracellular protein from microalgae with biological, mechanical and chemical approaches. In this study, based on comparison of different extraction methods, a new protocol was established to maximize extract amounts of protein, which was involved in ethanol soaking, enzyme digest, ultrasonication and homogenization techniques. Under the optimized conditions, 72.4% of protein was extracted from the microalgae Chlorella pyrenoidosa, which should contribute to the research and development of Chlorella protein in functional food and medicine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. 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.

  7. 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

  8. Regulation of myofibrillar accumulation in chick muscle cultures - Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

    Science.gov (United States)

    Silver, Geri; Etlinger, Joseph D.

    1985-01-01

    The effects of calcium on the synthesis and the degradation of individual myofibrillar proteins were investigated using primary chick-leg skeletal muscle cultures labeled with S-35-methionine (for protein accumulation experiments) or Ca(2+)-45 (for calcium efflux experiments). It was found that the turnover of individual contractile proteins is regulated nonuniformly by a calcium-dependent mechanism involving lysosomes. The results also indicate that contractile proteins are released from the myofibril before their breakdown to amino acids.

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

    Science.gov (United States)

    Xu, Kaijia; Wang, Yuzhi; Huang, Yanhua; Li, Na; Wen, Qian

    2015-03-15

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Identification of differentially accumulated proteins involved in regulating independent and combined osmosis and cadmium stress response in Brachypodium seedling roots.

    Science.gov (United States)

    Chen, Ziyan; Zhu, Dong; Wu, Jisu; Cheng, Zhiwei; Yan, Xing; Deng, Xiong; Yan, Yueming

    2018-05-17

    In this study, we aimed to identify differentially accumulated proteins (DAPs) involved in PEG mock osmotic stress, cadmium (Cd 2+ ) stress, and their combined stress responses in Brachypodium distachyon seedling roots. The results showed that combined PEG and Cd 2+ stresses had more significant effects on Brachypodium seedling root growth, physiological traits, and ultrastructures when compared with each individual stress. Totally, 106 DAPs were identified that are responsive to individual and combined stresses in roots. These DAPs were mainly involved in energy metabolism, detoxification and stress defense and protein metabolism. Principal component analysis revealed that DAPs from Cd 2+ and combined stress treatments were grouped closer than those from osmotic stress treatment, indicating that Cd 2+ and combined stresses had more severe influences on the root proteome than osmotic stress alone. Protein-protein interaction analyses highlighted a 14-3-3 centered sub-network that synergistically responded to osmotic and Cd 2+ stresses and their combined stresses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 14 key DAP genes revealed that most genes showed consistency between transcriptional and translational expression patterns. A putative pathway of proteome metabolic changes in Brachypodium seedling roots under different stresses was proposed, which revealed a complicated synergetic responsive network of plant roots to adverse environments.

  11. Estimation of extractable protein in botanical fractions of legume and grass species

    DEFF Research Database (Denmark)

    Solati, Zeinab; Jørgensen, Uffe; Eriksen, Jørgen

    2018-01-01

    With a globally strong interest in bio-based products such as fuels and chemicals, a feasible source of protein for the industry with positive economic impacts could be from leaves. However, more knowledge is needed on how to improve the content of extractable protein. Grasses and legumes have a ...

  12. Lack of Globulin Synthesis during Seed Development Alters Accumulation of Seed Storage Proteins in Rice

    Directory of Open Access Journals (Sweden)

    Hye-Jung Lee

    2015-06-01

    Full Text Available The major seed storage proteins (SSPs in rice seeds have been classified into three types, glutelins, prolamins, and globulin, and the proportion of each SSP varies. It has been shown in rice mutants that when either glutelins or prolamins are defective, the expression of another type of SSP is promoted to counterbalance the deficit. However, we observed reduced abundances of glutelins and prolamins in dry seeds of a globulin-deficient rice mutant (Glb-RNAi, which was generated with RNA interference (RNAi-induced suppression of globulin expression. The expression of the prolamin and glutelin subfamily genes was reduced in the immature seeds of Glb-RNAi lines compared with those in wild type. A proteomic analysis of Glb-RNAi seeds showed that the reductions in glutelin and prolamin were conserved at the protein level. The decreased pattern in glutelin was also significant in the presence of a reductant, suggesting that the polymerization of the glutelin proteins via intramolecular disulfide bonds could be interrupted in Glb-RNAi seeds. We also observed aberrant and loosely packed structures in the storage organelles of Glb-RNAi seeds, which may be attributable to the reductions in SSPs. In this study, we evaluated the role of rice globulin in seed development, showing that a deficiency in globulin could comprehensively reduce the expression of other SSPs.

  13. Evaluation of Aqueous Extracted Moringa Leaf Meal as a Protein ...

    African Journals Online (AJOL)

    saponins and to a lesser extent tannin, phytic acid and ... (III) oxide (BDH 277574Q) as an inert marker at an .... DP/DE ratio (mg DP/kJ DE) = Digestible protein/ ..... colour reaction with the reagents. .... hydrochloric acidic and calcium hydroxide.

  14. [PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

    Science.gov (United States)

    Sudarkina, O Yu; Dergunova, L V

    2015-01-01

    Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

  15. 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

  16. 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.

  17. 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.

  18. Buddleja officinalis Maximowicz extract inhibits lipid accumulation on adipocyte differentiation in 3T3-L1 cells and high-fat mice.

    Science.gov (United States)

    Roh, Changhyun; Park, Min-Kyoung; Shin, Hee-June; Jung, Uhee; Kim, Jin-Kyu

    2012-07-23

    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.

  19. Protein extraction from methanol fixed paraffin embedded tissue blocks: A new possibility using cell blocks

    Science.gov (United States)

    Kokkat, Theresa J.; McGarvey, Diane; Patel, Miral S.; Tieniber, Andrew D.; LiVolsi, Virginia A.; Baloch, Zubair W.

    2013-01-01

    Background: Methanol fixed and paraffin embedded (MFPE) cellblocks are an essential cytology preparation. However, MFPE cellblocks often contain limited material and their relatively small size has caused them to be overlooked in biomarker discovery. Advances in the field of molecular biotechnology have made it possible to extract proteins from formalin fixed and paraffin embedded (FFPE) tissue blocks. In contrast, there are no established methods for extracting proteins from MFPE cellblocks. We investigated commonly available CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) buffer, as well as two commercially available Qiagen® kits and compared their effectiveness on MFPE tissue for protein yields. Materials and Methods: MFPE blocks were made by Cellient™ automated system using human tissue specimens from normal and malignant specimens collected in ThinPrep™ Vials. Protein was extracted from Cellient-methanol fixed and paraffin embedded blocks with CHAPS buffer method as well as FFPE and Mammalian Qiagen® kits. Results: Comparison of protein yields demonstrated the effectiveness of various protein extraction methods on MFPE cellblocks. Conclusion: In the current era of minimally invasive techniques to obtain minimal amount of tissue for diagnostic and prognostic purposes, the use of commercial and lab made buffer on low weight MFPE scrapings obtained by Cellient® processor opens new possibilities for protein biomarker research. PMID:24403950

  20. 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

  1. 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.

  2. The extraction of liquid, protein molecules and yeast cells from paper through surface acoustic wave atomization.

    Science.gov (United States)

    Qi, Aisha; Yeo, Leslie; Friend, James; Ho, Jenny

    2010-02-21

    Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

  3. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    Science.gov (United States)

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. 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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts

    OpenAIRE

    Redmile-Gordon, M.A.; Armenise, E.; White, R.P.; Hirsch, P.R.; Goulding, K.W.T.

    2013-01-01

    Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colori...

  7. 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.

  8. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Protein-accumulating cells and dilated cisternae of the endoplasmic reticulum in three glucosinolate-containing genera: Armoracia, Capparis, Drypetes.

    Science.gov (United States)

    Jørgensen, L B; Behnke, H D; Mabry, T J

    1977-01-01

    Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former "myrosin cells") occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed.

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

    DEFF Research Database (Denmark)

    Bartels, Emil D; Nielsen, Jan M; Hellgren, Lars I

    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 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...

  11. Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

    Science.gov (United States)

    Huang, Tengfang; Jander, Georg

    2017-10-01

    Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

  12. 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.

  13. 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.

  14. 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.

  15. 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...

  16. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

    DEFF Research Database (Denmark)

    Weber, Daniela; Davies, Michael J.; Grune, Tilman

    2015-01-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple...

  17. Protein nativity explains emulsifying properties of aqueous extracted protein components from yellow pea

    NARCIS (Netherlands)

    Geerts, Marlies E.J.; Nikiforidis, Constantinos V.; Goot, van der Atze Jan; Padt, van der Albert

    2017-01-01

    In this paper, the emulsifying properties of a protein-enriched fraction from pea are unravelled. The emulsifying properties of mildly fractionated protein fractions from yellow pea and compared to those of commercial pea protein isolate. The emulsion stability of an oil-in-water emulsions were

  18. Comparison of the method of diffusive gels in thin films with conventional extraction techniques for evaluating zinc accumulation in plants and isopods

    International Nuclear Information System (INIS)

    Koster, Marijke; Reijnders, Lucas; Oost, Nathalie R. van; Peijnenburg, Willie J.G.M.

    2005-01-01

    The measurement of diffusive gels in thin films (DGT) has recently been developed to assess metal bioavailability in soils. The DGT-method is based on diffusion in a porous matrix. To test the predictive capabilities of the method with regard to metal bioavailability, a study was set up with 28 soils having a variety of textures and amounts of zinc salts added. Correlation and regression analyses were performed to compare DGT-extracted zinc levels to zinc concentrations obtained by extraction with 0.01 M CaCl 2 and 0.43 M HNO 3 , digestion with aqua regia and the zinc concentration in pore water. The amount of zinc extracted with CaCl 2 correlated well with DGT-extracted zinc levels in all soils spiked with different amounts of ZnCl 2 . A similar correlation was not found for zinc concentrations in soil samples collected in the field. Experiments were performed to compare zinc content in organisms and in soils. The organisms tested were plants (grass, lettuce and lupine) and the hard bodied soil dwelling isopod Oniscus asellus. Good correlations were found between zinc accumulation in grass and lettuce and the C E (effective concentration) measured by a DGT-device, CaCl 2 extracted zinc and the zinc content in the pore water of all soils. The correlation with C E was not significant for lupine, neither for spiked soils, nor for field soils (p ≤ 0.001). Zinc levels in the isopods were not significantly related to any set of zinc measurements. From a synthesis of all results obtained it is concluded that the DGT-methodology does not have an additional value in predicting bioavailability of zinc in terrestrial ecosystems as compared to conventional extraction methods. - Capsule: The newly developed method of diffusive gels in Thin films (DGT) does not have an added value over conventional extraction techniques in predicting zinc uptake by plants and isopods

  19. Dry matter yield, chemical composition and estimated extractable protein of legume and grass species during the spring growth

    DEFF Research Database (Denmark)

    Solati, Zeinab; Jørgensen, Uffe; Eriksen, Jørgen

    2017-01-01

    Carbohydrate and Protein System across six harvests during the spring growth. RESULTS The estimated extractable protein [g kg−1 dry matter (DM)] defined as the easily available fractions B1+B2 was significantly higher in white clover and lucerne at all harvests while, if the more cell wall attached fraction B3...... for protein production purpose in a biorefinery due to its high extractable protein content per kg DM. In order to maximise the protein production capacity, harvest should take place during early growth due to a decline in protein extractability with maturity. The final economy of the concept will depend...

  20. Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

    Science.gov (United States)

    Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

    2018-02-20

    Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

  1. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. 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.

  3. Locally accumulated extractable compounds in mycorrhizal parts of maize roots suppress the growth of hyphae of glomus intraradices

    Czech Academy of Sciences Publication Activity Database

    Gryndler, Milan; Hršelová, Hana; Malcová, Radka; Cajthaml, Tomáš; Halada, Petr

    2003-01-01

    Roč. 38, - (2003), s. 125-138 ISSN 0015-5551 R&D Projects: GA ČR GA526/97/0595 Institutional research plan: CEZ:AV0Z6005908; CEZ:AV0Z5020903 Keywords : fractions * patassium * root extract Subject RIV: EE - Microbiology, Virology Impact factor: 1.057, year: 2003

  4. 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 (Pmeatballs 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. 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.

  6. Extraction and comparison of proteins from natural rubber latex by conventional and ionizing radiation methods

    International Nuclear Information System (INIS)

    Rogero, Sizue O.; Spencer, Patrick J.; Campos, Vania E.; Lusvarghi, Fabio M.; Higa, Olga Z.

    1997-01-01

    Several proteins in natural rubber latex (NRL) have been assigned to be significant allergens. It is known that proteins submitted to ionizing radiation suffer denaturation and immunochemical modification resulting in low antigenic reactivity. The aim of this study was to extract and compare water extractable proteins from NRL films vulcanized by conventional and by ionizing radiation methods. SDS-polyacrylamide gel electrophoresis (SDS--PAGE) and high pressure liquid chromatography (HPLC) showed a diffuse protein band of about 14 KDa, which we believe is rubber elongation factor (REF), in both eluates, but smaller in latex film vulcanized by ionizing radiation. REF has been suggested to be a major latex allergen. These data suggest that ionizing radiation vulcanization could be an useful method for the production of NRL goods with low antigenicity. (author). 8 refs., 2 figs., 1 tab

  7. Global Picture of Protein Regulation in Response to Dibutyl Phthalate (DBP) Stress of Two Brassica parachinensis Cultivars Differing in DBP Accumulation.

    Science.gov (United States)

    Zhao, Hai-Ming; Huang, He-Biao; Du, Huan; Xiang, Lei; Mo, Ce-Hui; Li, Yan-Wen; Cai, Quan-Ying; Li, Hui; Liu, Jie-Sheng; Zhou, Dong-Mei; Wong, Ming-Hung

    2018-05-09

    iTRAQ analysis was used to map the proteomes of two Brassica parachinensis cultivars that differed in dibutyl phthalate (DBP) accumulation. A total of 5699 proteins were identified to obtain 152 differentially regulated proteins, of which 64 and 48 were specific to a high- and a low-DBP-accumulation cultivar, respectively. Genotype-specific biological processes were involved in coping with DBP stress, accounting for the variation in DBP tolerance and accumulation. Formation of high DBP accumulation in B. parachinensis might attribute to the more effective regulation of protein expression in physiology and metabolism, including (a) enhanced cell wall biosynthesis and modification, (b) better maintenance of photosynthesis and energy balance, (c) greatly improved total capacity for antioxidation and detoxification, and (d) enhanced cellular transport and signal transduction. Our novel findings contribute to a global picture of DBP-induced alterations of protein profiles in crops and provide valuable information for the development of molecular-assisted breeds of low-accumulation cultivars.

  8. The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Famin O.

    2003-03-01

    Full Text Available Ferriprotoporphyrin IX (FPIX is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleoside monophosphate kinase and pyrimidine 5'- nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-α, phoshoglycerate kinase, glyceraldehyde 3- phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6- phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

  9. Systematic identification of yeast proteins extracted into model wine during aging on the yeast lees.

    Science.gov (United States)

    Rowe, Jeffrey D; Harbertson, James F; Osborne, James P; Freitag, Michael; Lim, Juyun; Bakalinsky, Alan T

    2010-02-24

    Total protein and protein-associated mannan concentrations were measured, and individual proteins were identified during extraction into model wines over 9 months of aging on the yeast lees following completion of fermentations by seven wine strains of Saccharomyces cerevisiae. In aged wines, protein-associated mannan increased about 6-fold (+/-66%), while total protein only increased 2-fold (+/-20%), which resulted in a significantly greater protein-associated mannan/total protein ratio for three strains. A total of 219 proteins were identified among all wine samples taken over the entire time course. Of the 17 "long-lived" proteins detected in all 9 month samples, 13 were cell wall mannoproteins, and four were glycolytic enzymes. Most cytosolic proteins were not detected after 6 months. Native mannosylated yeast invertase was assayed for binding to wine tannin and was found to have a 10-fold lower affinity than nonglycosylated bovine serum albumin. Enrichment of mannoproteins in the aged model wines implies greater solution stability than other yeast proteins and the possibility that their contributions to wine quality may persist long after bottling.

  10. Arabinogalactan Proteins Accumulate in the Cell Walls of Searching Hyphae of the Stem Parasitic Plants, Cuscuta campestris and Cuscuta japonica.

    Science.gov (United States)

    Hozumi, Akitaka; Bera, Subhankar; Fujiwara, Daiki; Obayashi, Takeshi; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Aoki, Koh

    2017-11-01

    Stem parasitic plants (Cuscuta spp.) develop a specialized organ called a haustorium to penetrate their hosts' stem tissues. To reach the vascular tissues of the host plant, the haustorium needs to overcome the physical barrier of the cell wall, and the parasite-host interaction via the cell wall is a critical process. However, the cell wall components responsible for the establishment of parasitic connections have not yet been identified. In this study, we investigated the spatial distribution patterns of cell wall components at a parasitic interface using parasite-host complexes of Cuscuta campestris-Arabidopsis thaliana and Cuscuta japonica-Glycine max. We focused on arabinogalactan proteins (AGPs), because AGPs accumulate in the cell walls of searching hyphae of both C. campestris and C. japonica. We found more AGPs in elongated haustoria than in pre haustoria, indicating that AGP accumulation is developmentally regulated. Using in situ hybridization, we identified five genes in C. campestris that encode hyphal-expressed AGPs that belong to the fasciclin-like AGP (FLA) family, which were named CcFLA genes. Three of the five CcFLA genes were expressed in the holdfast, which develops on the Cuscuta stem epidermis at the attachment site for the host's stem epidermis. Our results suggest that AGPs are involved in hyphal elongation and adhesion to host cells, and in the adhesion between the epidermal tissues of Cuscuta and its host. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. 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.

  12. Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.

    Science.gov (United States)

    Rurek, Michal

    2010-08-18

    Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress. All the analyzed plant species showed constitutive accumulation of thermostable mitochondrial putative dehydrins ranging from 50 to 70 kDa. The mitochondrial dehydrin-like proteins observed in cauliflower and Arabidopsis ranged from 10 to 100 kDa and in lupin imbibed seeds and hypocotyls--from 20 to 90 kDa. Cold treatment increased mainly the accumulation of 10-100 kDa cauliflower and Arabidopsis dehydrin-like proteins, in the patterns different in cauliflower leaf and inflorescence mitochondria. However, in lupin mitochondria, cold affected mainly 25-50 kDa proteins and seemed to induce the appearance of some novel dehydrin-like proteins. The influence of frost stress on cauliflower leaf mitochondrial dehydrin- like proteins was less significant. The impact of heat stress was less significant in lupin and Arabidopsis than in cauliflower inflorescence mitochondria. Cauliflower mitochondrial dehydrin-like proteins are localized mostly in the mitochondrial matrix; it seems that some of them may interact with mitochondrial membranes. All the results reveal an unexpectedly broad spectrum of dehydrin-like proteins accumulated during some abiotic stress in the mitochondria of the plant species analyzed. They display only limited similarity in size to those reported previously in maize, wheat and rye

  13. 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...

  14. 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;

  15. Pressurized hot water extraction of proteins from Sambucus nigra L. branches

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Hohnová, Barbora

    2017-01-01

    Roč. 108, DEC (2017), s. 312-315 ISSN 0926-6690 Grant - others:GA AV ČR(CZ) R200311521 Institutional support: RVO:68081715 Keywords : elderberry * pressurized hot water extraction * proteins Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 3.181, year: 2016

  16. 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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

    We describe the formation of amorphous selenium (α-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO 3 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 3 2- ions to Se 0 , but also controls the nucleation and growth of Se 0 , 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 SeO 3 2- ions by Capsicum annuum L extract

  18. Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris

    Science.gov (United States)

    2013-01-01

    Background Failure of colony PCRs in green microalga Chlorella vulgaris is typically attributed to the difficulty in disrupting its notoriously rigid cell walls for releasing the genetic materials and therefore the development of an effective colony PCR procedure in C. vulgaris presents a challenge. Results Here we identified that colony PCR results were significantly affected by the accumulated lipids rather than the rigid cell walls of C. vulgaris. The higher lipids accumulated in C. vulgaris negatively affects the effective amplification by DNA polymerase. Based on these findings, we established a simple and extremely effective colony PCR procedure in C. vulgaris. By simply pipetting/votexing the pellets of C. vulgaris in 10 ul of either TE (10 mM Tris/1 mM EDTA) or 0.2% SDS buffer at room temperature, followed by the addition of 10 ul of either hexane or Phenol:Chloroform:Isoamyl Alcohol in the same PCR tube for extraction. The resulting aqueous phase was readily PCR-amplified as genomic DNA templates as demonstrated by successful amplification of the nuclear 18S rRNA and the chloroplast rbcL gene. This colony PCR protocol is effective and robust in C. vulgaris and also demonstrates its effectiveness in other Chlorella species. Conclusions The accumulated lipids rather than the rigid cell walls of C. vulgaris significantly impede the extraction of genetic materials and subsequently the effective colony PCRs. The finding has the potential to aid the isolation of high-quality total RNAs and mRNAs for transcriptomic studies in addition to the genomic DNA isolation in Chlorella. PMID:24219401

  19. 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

  20. 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.

  1. Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy.

    Science.gov (United States)

    Konuma, Tsuyoshi; Harada, Erisa; Sugase, Kenji

    2015-12-01

    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.

  2. 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.

  3. Increased accumulation of dendritic cells in celiac disease associates with increased expression of autophagy protein LC3

    Directory of Open Access Journals (Sweden)

    Paramaguru Rajaguru

    2013-01-01

    Full Text Available Background: Celiac disease (CD an immune-mediated disorder associates with accumulation of dendritic cell (DC in duodenal mucosa. Autophagy has recently been implicated in autoantigen formation. However, its role in CD is still unknown. Aim: To examine role of autophagic protein LC3 expressed by activated DC in CD. Materials and Methods : Thirty CD patients were analyzed at initial presentation and after 6 months of gluten-free diet (GFD. Duodenal biopsies were studied for histological changes and CD11c, CD86, and MAP1LC3A expressions by double immunohistochemistry (IHC. Masson′s trichrome (MT staining was used to assess basement membrane (BM thickness and Oil Red O (ORO staining for mucosal lipid deposit. Polymerase chain reaction (PCR was performed for HLA-DQ system. Statistical analysis was done using paired and unpaired t test, chi-square test, Fisher′s exact test, and McNemar-Bowker test. A P-value <0.05 was considered statistically significant. Results: HLA-DQ2 and HLA-DQ8 alleles were present in all studied patients. Increased BM thickness was observed in 63% and 73% had ORO-positive lipid in surface lining epithelium. Pre-treatment biopsies showed increased DCs expressing LC3, which were significantly less in follow-up biopsies. The follow-up biopsies had shown significant reduction in BM thickness and ORO. Conclusion : Histological improvement in duodenal biopsies was associated with reduction in activated DCs expressing autophagic protein, which probably play important role in pathogenesis of an autoimmune disorder like CD.

  4. 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.

  5. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    International Nuclear Information System (INIS)

    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 (IC 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 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 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

  6. 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

  7. Alkali solution extraction of rice residue protein isolates: Influence of alkali concentration on protein functional, structural properties and lysinoalanine formation.

    Science.gov (United States)

    Hou, Furong; Ding, Wenhui; Qu, Wenjuan; Oladejo, Ayobami Olayemi; Xiong, Feng; Zhang, Weiwei; He, Ronghai; Ma, Haile

    2017-03-01

    This study evaluated the nutrient property and safety of the rice residue protein isolates (RRPI) product (extracted by different alkali concentrations) by exploring the protein functional, structural properties and lysinoalanine (LAL) formation. The results showed that with the rising of alkali concentration from 0.03M to 0.15M, the solubility, emulsifying and foaming properties of RRPI increased at first and then descended. When the alkali concentration was greater than 0.03M, the RRPI surface hydrophobicity decreased and the content of thiol and disulfide bond, Lys and Cys significantly reduced. By the analysis of HPLC, the content of LAL rose up from 276.08 to 15,198.07mg/kg and decreased to 1340.98mg/kg crude protein when the alkali concentration increased from 0.03 to 0.09M and until to 0.15M. These results indicated that RRPI alkaline extraction concentration above 0.03M may cause severe nutrient or safety problems of protein. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. 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.

  9. Overexpression of Cholesteryl Ester Transfer Protein Increases Macrophage-Derived Foam Cell Accumulation in Atherosclerotic Lesions of Transgenic Rabbits

    Directory of Open Access Journals (Sweden)

    Shoucui Gao

    2017-01-01

    Full Text Available High levels of plasma high-density lipoprotein-cholesterol (HDL-C are inversely associated with the risk of atherosclerosis and other cardiovascular diseases; thus, pharmacological inhibition of cholesteryl ester transfer protein (CETP is considered to be a therapeutic method of raising HDL-C levels. However, many CETP inhibitors have failed to achieve a clinical benefit despite raising HDL-C. In the study, we generated transgenic (Tg rabbits that overexpressed the human CETP gene to examine the influence of CETP on the development of atherosclerosis. Both Tg rabbits and their non-Tg littermates were fed a high cholesterol diet for 16 weeks. Plasma lipids and body weight were measured every 4 weeks. Gross lesion areas of the aortic atherosclerosis along with lesional cellular components were quantitatively analyzed. Overexpression of human CETP did not significantly alter the gross atherosclerotic lesion area, but the number of macrophages in lesions was significantly increased. Overexpression of human CETP did not change the plasma levels of total cholesterol or low-density lipoprotein cholesterol but lowered plasma HDL-C and increased triglycerides. These data revealed that human CETP may play an important role in the development of atherosclerosis mainly by decreasing HDL-C levels and increasing the accumulation of macrophage-derived foam cells.

  10. 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.

  11. An Extraordinary Accumulation of (-)-Pinoresinol in Cell-Free Extracts of Forsythia intermedia: Evidence for Enantiospecific Reduction of (+)-Pinoresinol

    Science.gov (United States)

    Katayama, Takeshi; Davin, Laurence B.; Lewis, Norman G.

    1992-01-01

    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 catalysis 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 greater than 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 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.

  12. Structural classification of proteins using texture descriptors extracted from the cellular automata image.

    Science.gov (United States)

    Kavianpour, Hamidreza; Vasighi, Mahdi

    2017-02-01

    Nowadays, having knowledge about cellular attributes of proteins has an important role in pharmacy, medical science and molecular biology. These attributes are closely correlated with the function and three-dimensional structure of proteins. Knowledge of protein structural class is used by various methods for better understanding the protein functionality and folding patterns. Computational methods and intelligence systems can have an important role in performing structural classification of proteins. Most of protein sequences are saved in databanks as characters and strings and a numerical representation is essential for applying machine learning methods. In this work, a binary representation of protein sequences is introduced based on reduced amino acids alphabets according to surrounding hydrophobicity index. Many important features which are hidden in these long binary sequences can be clearly displayed through their cellular automata images. The extracted features from these images are used to build a classification model by support vector machine. Comparing to previous studies on the several benchmark datasets, the promising classification rates obtained by tenfold cross-validation imply that the current approach can help in revealing some inherent features deeply hidden in protein sequences and improve the quality of predicting protein structural class.

  13. 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...

  14. Protein Extractability

    African Journals Online (AJOL)

    (Council of Scientific and Industrial Research), Mysore 570 020, India. 2Department of Agricultural Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria. (Received: ... Ife Journal of Science vol. ..... Conference, Kathmandu, Nepal, Aug. 30.

  15. Valorisation of Jatropha curcas: Solubilisation of proteins and sugars from the NaOH extracted de-oiled press cake

    NARCIS (Netherlands)

    Kootstra, A.M.J.; Beeftink, H.H.; Sanders, J.P.M.

    2011-01-01

    In this study, we investigated the possibilities for increasing the valorisation of de-oiled Jatropha press cake (DO-JPC). The studied raw material is the by-product of the alkaline protein extraction of the DO-JPC: NaOH Extracted DO-JPC (NEDO-JPC). Protein solubilisation of NEDO-JPC was performed

  16. 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

    2013-10-09

    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.

  17. Comparison of the method of diffusive gels in thin films with conventional extraction techniques for evaluating zinc accumulation in plants and isopods

    Energy Technology Data Exchange (ETDEWEB)

    Koster, Marijke [National Institute of Public Health and the Environment, Laboratory for Ecological Risk Assessment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Open University Netherlands, Heerlen (Netherlands); Reijnders, Lucas [Open University Netherlands, Heerlen (Netherlands); Oost, Nathalie R. van [National Institute of Public Health and the Environment, Laboratory for Ecological Risk Assessment, P.O. Box 1, 3720 BA Bilthoven (Netherlands); Peijnenburg, Willie J.G.M. [National Institute of Public Health and the Environment, Laboratory for Ecological Risk Assessment, P.O. Box 1, 3720 BA Bilthoven (Netherlands)]. E-mail: wjgm.peijnenburg@rivm.nl

    2005-01-01

    The measurement of diffusive gels in thin films (DGT) has recently been developed to assess metal bioavailability in soils. The DGT-method is based on diffusion in a porous matrix. To test the predictive capabilities of the method with regard to metal bioavailability, a study was set up with 28 soils having a variety of textures and amounts of zinc salts added. Correlation and regression analyses were performed to compare DGT-extracted zinc levels to zinc concentrations obtained by extraction with 0.01 M CaCl{sub 2} and 0.43 M HNO{sub 3}, digestion with aqua regia and the zinc concentration in pore water. The amount of zinc extracted with CaCl{sub 2} correlated well with DGT-extracted zinc levels in all soils spiked with different amounts of ZnCl{sub 2}. A similar correlation was not found for zinc concentrations in soil samples collected in the field. Experiments were performed to compare zinc content in organisms and in soils. The organisms tested were plants (grass, lettuce and lupine) and the hard bodied soil dwelling isopod Oniscus asellus. Good correlations were found between zinc accumulation in grass and lettuce and the C{sub E} (effective concentration) measured by a DGT-device, CaCl{sub 2} extracted zinc and the zinc content in the pore water of all soils. The correlation with C{sub E} was not significant for lupine, neither for spiked soils, nor for field soils (p {<=} 0.001). Zinc levels in the isopods were not significantly related to any set of zinc measurements. From a synthesis of all results obtained it is concluded that the DGT-methodology does not have an additional value in predicting bioavailability of zinc in terrestrial ecosystems as compared to conventional extraction methods. - Capsule: The newly developed method of diffusive gels in Thin films (DGT) does not have an added value over conventional extraction techniques in predicting zinc uptake by plants and isopods.

  18. 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

  19. 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 maturation.

  20. Control of protein synthesis in cell-free extracts of sea urchin embryos

    International Nuclear Information System (INIS)

    Hansen, L.J.; Huang, W.I.; Jagus, R.

    1986-01-01

    Although the increase in protein synthesis that occurs after fertilization of sea urchin eggs results from increased utilization of stored maternal mRNA, the underlying mechanism is unknown. The authors have prepared cell-free extracts from S.purpuratus and A.puctulata unfertilized eggs and 2-cell embryos that retain the protein synthetic differences observed in vivo. The method is based on that of Dr. Alina Lopo. 35 S methionine incorporation is linear during a 30 min incubation and is 10-20 fold higher in extracts from 2-cell embryos than unfertilized eggs. Addition of purified mRNA does not stimulate these systems, suggesting a regulatory mechanism other than mRNA masking. Addition of rabbit reticulocyte ribosomal salt wash stimulated protein synthesis in extracts from eggs but not embryos, suggesting deficiencies in translational components in unfertilized eggs. Mixing of egg and embryo lysates indicated the presence of a weak protein synthesis inhibitor in eggs. Translational control in developing sea urchin embryos thus appears to be complex, involving both stimulatory and inhibitory factors

  1. RESEARCH METHODOLOGY OF SUS SCROFA TISSUE EXTRACTS PROTEIN-PEPTIDE COMPONENTS

    Directory of Open Access Journals (Sweden)

    Ekaterina R.. Vasilevskaya

    2017-01-01

    Full Text Available The article presents a comparative analysis of four methods for quantifying the protein-peptide complexes content in extracts obtained from animal raw materials, as well as the low- and highmolecular weight extract fractions: the direct spectrophotometric determination at wavelengths of 260 and 280 nm with subsequent calculation by the Kalckar formula; the biuret reaction by the Kingsley-Weichselbaum method; the method with Bradford reagent and the standard Lowry method. Experimental data analysis demonstrates that in case of the extract that contains protein-peptidic complexes in different molecular weights range, the Kingsley-Weichselbaum method shows the highest quality of protein concentration determination; while studying highmolecular weight fraction (more than 30 kDa, it is possible to obtain more information by combining the spectrophotometric method and the Kingsley-Weichselbaum method. Low-molecular weight fractions (less than 30 kD should be investigated by complex methods including the spectrophotometric method, Lowry and Bradford methods. These methods make it possible to presumably estimate protein molecules size ranges (by amount of peptide bonds, and also to determine hydrophobic and aromatic amino acids presence.

  2. Extraction methods and test techniques for detection of vegetable proteins in meat products. I. Qualitative detection of soya derivatives.

    Science.gov (United States)

    Hyslop, N S

    1976-06-01

    Extracts of 3 soya bean preparations, used commercially in certain countries to replace part of the meat in popular meat products, were made by treatment with (i) sodium dodecyl sulphate, (ii) Triton-X100 or (iii) n-Butanol. Similar extracts were made from beef and pork. All extracts were examined by electrophoretic and immunological techniques. Stained polyacrylamide gels revealed distinctive protein bands after electrophoresis. The migration rates of corresponding bands differed between beef and pork extracts. However, the migration rates of vegetable bands revealed certain similarities, but differed very greatly from those of animal origin. Characteristic fast-migrating S-bands were distinguishable only in extracts of vegetable protein. Immunodiffusion tests, using antisera produced in rabbits against each extract, revealed varying degrees of similarity between extracts of vegetable origin, but the antisera were specific for either vegetable or animal protein.

  3. 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). Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Clarkson, Claudia; Mirosa, Miranda; Birch, John

    2018-02-09

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

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

    Directory of Open Access Journals (Sweden)

    Claudia Clarkson

    2018-02-01

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

  6. Radiobiological long-term accumulation of environmental alpha radioactivity in extracted human teeth and animal bones in Malaysia

    International Nuclear Information System (INIS)

    Almayahi, B.A.; Tajuddin, A.A.; Jaafar, M.S.

    2014-01-01

    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). - Highlights: • Alpha emission rates in teeth from smokers slightly higher than non-smokers. • Difference between alpha rates in male and female tooth not statistically significant. • Alpha particles have the same effect at any age. • Difference between alpha rates in bones was statistically significant

  7. The relationship between lipid accumulation product, visceral adiposity index and high-sensitivity C-reactive protein in healthy adults

    Directory of Open Access Journals (Sweden)

    Yan ZHANG

    2013-03-01

    Full Text Available Objective  To investigate the correlation between lipid accumulation product (LAP, visceral adiposity index (VAI and high-sensitivity C-reactive protein (hs-CRP in adults, and explore whether to use such correlation as indications is superior to the traditional body fat index based on body mass index (BMI, waist circumference (WC, waist-hip ratio (WHR and waist-height ratio (WHtR. Methods  The present work was a cross-sectional study involving 501 healthy adults (321 males and 180 females from the community of Chongqing Municipality. Anthropometric indexes [height, weight, WC, hip circumference (HC], blood pressure (BP, fasting lipid profile and levels of fasting and post-load glucose, insulin and hs-CRP were measured, and BMI, WHR, WHtR, fasting insulin resistant homeostasis model assessment (HOMA-IR, LAP and VAI were calculated. The correlations between hs-CRP and other variables were analyzed. Results  Following the elevation of titer of the hs-CRP, LAP, VAI, BMI, WC, WHR, WHtR, BP, glucose level, HOMA-IR, insulin, triglyceride (TG, low-density lipoprotein cholesterol (LDL-C and apolipoprotein B (ApoB increased (P<0.05, while high-density lipoprotein cholesterol (HDL-C and apolipoprotein A1 (ApoA1 levels declined (P<0.0001. Pearson's correlation analysis demonstrated that hs-CRP was correlated with all variances (P<0.01 except for total cholesterol (TC (P=0.181 and LDL -C (P=0.325. According to forward stepwise multiple regression analysis with hs-CRP as the dependent variance, WC was the only variance entering the regression model. Conclusion  LAP, VAI levels are correlated with hs-CRP level but not the major determinant factors of hs-CRP. WC is stronger than other variances in the association with hs-CRP in adults, and is still an independent predictor of inflammation.

  8. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    Directory of Open Access Journals (Sweden)

    Adriana FIORINI

    2016-01-01

    Full Text Available Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC and sub-minimal inhibitory concentration (sub-MIC of the butanolic extract (BUTE of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1, amino acid metabolism (ILV5, PDC11 and protein synthesis (ASC1 pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides, it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

  9. Metabolic Regulation of CaMKII Protein and Caspases in Xenopus laevis Egg Extracts*

    Science.gov (United States)

    McCoy, Francis; Darbandi, Rashid; Chen, Si-Ing; Eckard, Laura; Dodd, Keela; Jones, Kelly; Baucum, Anthony J.; Gibbons, Jennifer A.; Lin, Sue-Hwa; Colbran, Roger J.; Nutt, Leta K.

    2013-01-01

    The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways. PMID:23400775

  10. Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

    Science.gov (United States)

    Kim, K H; Hemenway, C

    1997-05-26

    The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

  11. Accumulation of dry matter and nitrogen in the developing seeds of high protein mutant lines of Triticum Aestivum (L.) produced by the IAEA

    International Nuclear Information System (INIS)

    Mir Ali, N.; Nabulsi, I.

    1993-03-01

    Accumulation patterns of dry matter and nitrogen in the developing seeds of nine mutant lines produced by the IAEA and their mother Triticum Aestivum (L.) line were studied. The experiments lasted 2 years under rain fed conditions. Significant differences were found among the lines in dry matter and nitrogen rates, and periods of accumulation, whereas no significant differences were found in the final seed weight of the lines. The highest rates of accumulation for dry matter and nitrogen were accompanied with the shortest period of accumulation in two late flowering mutant lines. However, these two lines were the lowest in their yield per plot. The other mutant lines achieved the high nitrogen percentage in their seeds through the relative reduction in dry matter accumulation rate compared to their mother line rather than through higher rate of nitrogen accumulation. This study revealed some of the potential reasons behind the higher percentage of protein in the seeds of the mutant lines under investigation. (author). 17 refs., 3 figs., 2 tabs

  12. Effect of nigella sativa seeds extract on serum c-reactive protein in albino rats

    International Nuclear Information System (INIS)

    Bashir, M.U.; Qureshi, H.

    2014-01-01

    C-reactive protein (CRP) is an acute phase protein. It predicts future risk of cardiovascular diseases. Different medicinal plants and their active ingredients possess the ability to reduce serum CRP levels and hence inflammatory disorders and cardiovascular diseases. In our study, ethanolic extract of Nigella sativa seeds was evaluated in albino rats for its possible effect on serum CRP levels. Objective: The objective of this study was to determine the effect of ethanolic extract of Nigella sativa seeds on an acute inflammatory biomarker/mediator, C-reactive protein (CRP) in albino rats. Study Design: Randomized controlled trial (RCT). Place and Duration of Study: Physiology Department, Services Institute of Medical Sciences (SIMS), Lahore; from September to November, 2009. Subjects and Methods: The study was carried out on 90 male albino rats. Five percent (5%) formalin in a dose of 50 meu1 was injected into sub-plantar surface of right hind paw of each rat to produce inflammation. The rats were randomly divided into three groups of thirty each. Group A was given normal saline (control); group B was given Nigella sativa seed extract; and group C received diclofenac sodium, as a reference drug. CRP levels in each group were measured from blood samples taken 25 hours after giving formalin. Results: The ethanolic extract of Nigella sativa seeds, given intraperitoneally, caused highly significant (p<0.001) reduction in serum CRP levels as compared to control group. The reduction in CRP levels by ethanolic extract of Nigella sativa was also significantly (p<0.05) more than that produced by diclofenac sodium. Conclusion: Our results suggest that Nigella sativa possesses ability to reduce serum CRP levels significantly, after production of artificial inflammation, in albino rats. (author)

  13. Using co-occurrence network structure to extract synonymous gene and protein names from MEDLINE abstracts

    Directory of Open Access Journals (Sweden)

    Spackman K

    2005-04-01

    Full Text Available Abstract Background Text-mining can assist biomedical researchers in reducing information overload by extracting useful knowledge from large collections of text. We developed a novel text-mining method based on analyzing the network structure created by symbol co-occurrences as a way to extend the capabilities of knowledge extraction. The method was applied to the task of automatic gene and protein name synonym extraction. Results Performance was measured on a test set consisting of about 50,000 abstracts from one year of MEDLINE. Synonyms retrieved from curated genomics databases were used as a gold standard. The system obtained a maximum F-score of 22.21% (23.18% precision and 21.36% recall, with high efficiency in the use of seed pairs. Conclusion The method performs comparably with other studied methods, does not rely on sophisticated named-entity recognition, and requires little initial seed knowledge.

  14. Stability of spray-dried beetroot extract using oligosaccharides and whey proteins.

    Science.gov (United States)

    Carmo, Eloá Lourenço do; Teodoro, Rhana Amanda Ribeiro; Félix, Pedro Henrique Campelo; Fernandes, Regiane Victória de Barros; Oliveira, Érica Resende de; Veiga, Taís Regina Lima Abreu; Borges, Soraia Vilela; Botrel, Diego Alvarenga

    2018-05-30

    The properties and stability of spray-dried beetroot extract using maltodextrin (MD), inulin (IN), and whey protein isolate (WPI) as carrier agents were evaluated. The values of moisture, betalains content, and retention were 3.33-4.24%, 348.79-385.47 mg/100 g (dry-basis), and 88.45-95.69%, respectively. Higher values of antioxidant activity were observed for the treatments using WPI. The treatment with inulin alone presented higher hygroscopicity in the moisture adsorption isotherms at 25 °C and lower thermal stability when evaluating the thermogravimetric curves. When stored at 60 °C, the use of WPI alone conferred lower stability to the beetroot extract powder. In general, the simultaneous use of IN and WPI as carrier agents resulted in good stability of the beetroot extract powder, representing an opportunity for innovation in food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Isolation of proteins from ejaculated and capacitated sperms by various extraction approaches

    Czech Academy of Sciences Publication Activity Database

    Zigo, Michal; Jonáková, Věra; Postlerová, Pavla

    2010-01-01

    Roč. 33, Supplement 1 (2010), s. 68-68 ISSN 0105-6263. [6th European Congress of And rology. 29.09.2010-01.10.2010, Athens] R&D Projects: GA ČR(CZ) GA303/09/1285; GA MŠk(CZ) 1M06011; GA MZd NS10009-4/2008 Institutional research plan: CEZ:AV0Z50520701 Keywords : spermatozoa * protein extraction * 2D electrophoresis * zona pellucida * glycoproteins Subject RIV: CE - Biochemistry

  16. 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}.

  17. Boosting TAG Accumulation with Improved Biodiesel Production from Novel Oleaginous Microalgae Scenedesmus sp. IITRIND2 Utilizing Waste Sugarcane Bagasse Aqueous Extract (SBAE).

    Science.gov (United States)

    Arora, Neha; Patel, Alok; Pruthi, Parul A; Pruthi, Vikas

    2016-09-01

    This investigation utilized sugarcane bagasse aqueous extract (SBAE), a nontoxic, cost-effective medium to boost triacylglycerol (TAG) accumulation in novel fresh water microalgal isolate Scenedesmus sp. IITRIND2. Maximum lipid productivity of 112 ± 5.2 mg/L/day was recorded in microalgae grown in SBAE compared to modified BBM (26 ± 3 %). Carotenoid to chlorophyll ratio was 12.5 ± 2 % higher than in photoautotrophic control, indicating an increase in photosystem II activity, thereby increasing growth rate. Fatty acid methyl ester (FAME) profile revealed presence of C14:0 (2.29 %), C16:0 (15.99 %), C16:2 (4.05 %), C18:0 (3.41 %), C18:1 (41.55 %), C18:2 (12.41), and C20:0 (1.21 %) as the major fatty acids. Cetane number (64.03), cold filter plugging property (-1.05 °C), and oxidative stability (12.03 h) indicated quality biodiesel abiding by ASTM D6751 and EN 14214 fuel standards. Results consolidate the candidature of novel freshwater microalgal isolate Scenedesmus sp. IITRIND2 cultivated in SBAE, aqueous extract made from copious, agricultural waste sugarcane bagasse to increase the lipid productivity, and could further be utilized for cost-effective biodiesel production.

  18. Replacing soybean meal with gelatin extracted from cow skin and corn protein concentrate as a protein source in broiler diets.

    Science.gov (United States)

    Khalaji, S; Manafi, M; Olfati, Z; Hedyati, M; Latifi, M; Veysi, A

    2016-02-01

    Two experiments were conducted to investigate the effects of replacing soybean meal with gelatin extracted from cow skin and corn protein concentrate as a protein source in broiler diets. Experiments were carried out as a completely randomized design where each experiment involved 4 treatments of 6 replicates and 10 chicks in each pen. Soybean meal proteins in a corn-soy control diet were replaced with 15, 30, and 45% of cow skin gelatin (CSG) or corn protein concentrate (CPC), respectively, in experiments 1 and 2. BW and cumulative feed intake were measured at 7, 21, and 42 d of age. Blood characteristics, relative organs weight and length, ileal digesta viscosity, ileal morphology, and cecal coliform and Salmonella population were measured at 42 d of age. Apparent total tract digestibility of protein was determined during 35 to 42 d of age. Replacement of soybean meal with CSG severely inhibited BW gain, decreased feed intake, and increased FCR in broilers during the experimental period (P ≤ 0.01). The inclusion of CPC reduced BW and increased FCR significantly (P ≤ 0.05) at 21 and 42 d of age without any consequence in feed intake. Protein digestibility was reduced and ileal digesta viscosity was increased linearly by increasing the amount of CSG and CPC in the control diet (P ≤ 0.01). Replacement of soybean meal with CSG and CPC did not significantly alter blood cell profile and plasma phosphorus, creatinine, blood urea nitrogen, Aspartate transaminase, and HDL and LDL cholesterol concentration. The inclusion of CSG linearly (P ≤ 0.05) increased plasma uric acid concentration and alkaline phosphatase activity. Triglyceride and cholesterol levels were decreased significantly (P ≤ 0.05) when the amount of CSG replacement was 15%. The results of this experiment showed that using CSG and CPC negatively affects broiler performance and therefore is not a suitable alternative to soybean meal in commercial diets. © 2015 Poultry Science Association Inc.

  19. The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins.

    Science.gov (United States)

    López-Carrasco, Amparo; Flores, Ricardo

    2017-07-01

    Avocado sunblotch viroid (ASBVd), the type member of the family Avsunviroidae, replicates and accumulates in chloroplasts. Whether this minimal non-protein-coding circular RNA of 246-250 nt exists in vivo as a free nucleic acid or closely associated with host proteins remains unknown. To tackle this issue, the secondary structures of the monomeric circular (mc) (+) and (-) strands of ASBVd have been examined in silico by searching those of minimal free energy, and in vitro at single-nucleotide resolution by selective 2'-hydroxyl acylation analysed by primer extension (SHAPE). Both approaches resulted in predominant rod-like secondary structures without tertiary interactions, with the mc (+) RNA being more compact than its (-) counterpart as revealed by non-denaturing polyacryamide gel electrophoresis. Moreover, in vivo SHAPE showed that the mc ASBVd (+) form accumulates in avocado leaves as a free RNA adopting a similar rod-shaped conformation unprotected by tightly bound host proteins. Hence, the mc ASBVd (+) RNA behaves in planta like the previously studied mc (+) RNA of potato spindle tuber viroid, the type member of nuclear viroids (family Pospiviroidae), indicating that two different viroids replicating and accumulating in distinct subcellular compartments, have converged into a common structural solution. Circularity and compact secondary structures confer to these RNAs, and probably to all viroids, the intrinsic stability needed to survive in their natural habitats. However, in vivo SHAPE has not revealed the (possibly transient or loose) interactions of the mc ASBVd (+) RNA with two host proteins observed previously by UV irradiation of infected avocado leaves.

  20. Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

    Directory of Open Access Journals (Sweden)

    Erhui Xiong

    Full Text Available The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1 is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize and dicot (soybean and pea seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.

  1. Synthetic cold-inducible promoter enhances recombinant protein accumulation during Agrobacterium-mediated transient expression in Nicotiana excelsior at chilling temperatures.

    Science.gov (United States)

    Gerasymenko, I M; Sheludko, Y V

    2017-07-01

    To exploit cold-inducible biochemical processes beneficial for foreign mRNA transcription, translation and storage, as well as protein product stability, during Agrobacterium-mediated transient expression. The efficiency of three different 5'-regulatory sequences to achieve transient expression of the GFP-based reporter gene under chilling conditions (6-8 °C since the 3rd day post inoculation) was compared. We studied the upstream sequences of a cold-inducible Arabidopsis thaliana cor15a gene, the core element of 35S CaMV promoter fused to the TMV omega 5'-UTR, and the synthetic promoter including the 35S core sequence and two binding sites for cold-inducible CBF transcription factors (P_DRE::35S). Cultivation of plants transiently expressing reporter gene under control of the synthetic P_DRE::35S promoter under chilling conditions since the 3rd dpi led to the reliably higher reporter accumulation as compared to the other tested regulatory sequences under chilling or greenhouse conditions. Reporter protein fluorescence under chilling conditions using P_DRE::35S reached 160% as compared to the transient expression in the greenhouse. Period of transient expression considerably extended if plants were cultivated at chilling temperature since the 3rd dpi: reporter protein fluorescence reached its maximum at the 20th dpi and was detected in leaves up to the 65th dpi. The enhanced protein accumulation at low temperature was accompanied by the prolonged period of corresponding mRNA accumulation. Transient expression under chilling conditions using synthetic cold-inducible promoter enhances target protein accumulation and may decrease greenhouse heating expenses.

  2. High-protein diets prevent steatosis and induce hepatic accumulation of monomethyl branched-chain fatty acids

    NARCIS (Netherlands)

    Garcia Caraballo, Sonia C.; Comhair, Tine M.; Houten, Sander M.; Dejong, Cornelis H. C.; Lamers, Wouter H.; Koehler, S. Eleonore

    2014-01-01

    The hallmark of nonalcoholic fatty liver disease is steatosis of unknown etiology. To test how dietary protein decreases steatosis, we fed female C57BL/6 J mice low-fat (8 en%) or high-fat (42 en%) combined with low-protein (11 en%), high-protein (HP; 35 en%) or extra-high-protein (HPX; 58 en%)

  3. A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

    Science.gov (United States)

    Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

    2013-12-01

    Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

  4. Improving protein fold recognition by extracting fold-specific features from predicted residue-residue contacts.

    Science.gov (United States)

    Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

    2017-12-01

    Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent

  5. Phytotoxic mechanisms of bur cucumber seed extracts on lettuce with special reference to analysis of chloroplast proteins, phytohormones, and nutritional elements.

    Science.gov (United States)

    Lee, Seok-Min; Radhakrishnan, Ramalingam; Kang, Sang-Mo; Kim, Jin-Hyo; Lee, In-Yong; Moon, Bong-Kyu; Yoon, Byung-Wook; Lee, In-Jung

    2015-12-01

    Bioherbicides from plant extracts are an effective and environmentally friendly method to prevent weed growth. The present investigation was aimed at determining the inhibitory effect of bur cucumber seed extracts (BSE) on lettuce plant growth. Bur cucumber seeds were ground with water, and two different concentrations of seed extracts (10% and 20%) were prepared and applied to lettuce plants. Decreased plant height, number of leaves, leaf length, leaf width, anProd. Type: FTPd leaf area were found in lettuce exposed to BSE as compared with controls. A significant reduction in lettuce biomass was observed in 20% BSE-treated plants due to the presence of higher amounts of phenolic content in the extracts. Moreover, a significant inhibitory chemical, 2-linoleoyl glycerol, was identified in BSE extracts. The mechanism of plant growth inhibition was assayed in lettuce proteins by 2-dimensional electrophoresis (2-DE) and the LC-MS/MS method. In total, 57 protein spots were detected in plants treated with 20% BSE and control plants. Among these, 39 proteins were down-regulated and 18 proteins were up-regulated in plants exposed to 20% BSE as compared with controls. The presence of low levels of chlorophyll a/b binding protein and oxygen-evolving enhancer protein 1 in BSE-exposed plants reduced photosynthetic pigment synthesis and might be a reason for stunted plant growth. Indeed, the plant-growth stimulating hormone gibberellin was inhibited, and synthesis of stress hormones such as abscisic acid, jasmonic acid, and salicylic acid were triggered in lettuce by the effects of BSE. Uptake of essential nutrients, Ca, Fe, Mg, K, S, and Mo, was deficient and accumulation of the toxic ions Cu, Zn, and Na was higher in BSE-treated plants. The results of this study suggest that extracts of bur cucumber seeds can be an effective eco-friendly bioherbicide for weed control that work by inhibiting mechanisms of photosynthesis and regulating phytohormones and nutritional elements

  6. The anti-cancer property of proteins extracted from Gynura procumbens (Lour. Merr.

    Directory of Open Access Journals (Sweden)

    Chaw-Sen Hew

    Full Text Available Gynura procumbens (Lour. Merr. belongs to the Asteraceae Family. The plant is a well-known traditional herb in South East Asia and it is widely used to treat inflammation, kidney discomfort, high cholesterol level, diabetic, cancer and high blood pressure. Our earlier study showed the presence of valuable plant defense proteins, such as peroxidase, thaumatin-like proteins and miraculin in the leaf of G. procumbens. However, the effects of these defense proteins on cancers have never been determined previously. In the present study, we investigated the bioactivity of gel filtration fractionated proteins of G. procumbens leaf extract. The active protein fraction, SN-F11/12, was found to inhibit the growth of a breast cancer cell line, MDA-MB-231, at an EC50 value of 3.8 µg/mL. The mRNA expressions of proliferation markers, Ki67 and PCNA, were reduced significantly in the MDA-MB-23 cells treated with SN-F11/12. The expression of invasion marker, CCL2, was also found reduced in the treated MDA-MB-231 cells. All these findings highlight the anti-cancer property of SN-F11/12, therefore, the proteins in this fraction can be a potential chemotherapeutic agent for breast cancer treatment.

  7. Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato

    International Nuclear Information System (INIS)

    Oelmueller, R.; Kendrick, R.E.; Briggs, W.R.

    1989-01-01

    Polyclonal antibodies against pea phytochrome detect 2 protein bands (about 116 and 120 kDa) on blots of crude protein extracts and protein of microsomal preparations of dark-grown tomato seedlings. Both protein bands are undetectable in Western blots of the aurea mutant extracts. Neither protein band is detectable after isogenic wild-type seedlings are illuminated with 3 h of red light, either in the crude extract or in the membrane fraction of the irradiated seedlings; this result is consistent with the hypothesis that both bands are phytochrome. When dark-grown wild-type seedlings are illuminated with 3 h of red light or blue light against a red light background, the transcript levels for chlorophyll a/b-binding proteins of photosystem I and II, plastocyanin, and the subunit II of photosystem I increase. In all cases, the same fluence rate of blue light is much more effective than red light alone, a result that indicates the involvement of a blue/UV-A light photoreceptor in addition to the involvement of the far-red-absorbing form of phytochrome, Pfr. The aurea mutant responds neither to red light nor to blue light. Thus, no Pfr-independent induction of the four transcripts by a blue/UV-A light photoreceptor can be measured in the aurea mutant

  8. [FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS].

    Science.gov (United States)

    Wang, Xiaoyan; Chen, Ying; Huang, Yunchao; Zhou, Youquan; Zhao, Guangqiang; Ye, Lianhua; Lei, Yujie; Tang, Qi

    2015-01-01

    To explore the function of intercellular adhesion A (icaA), fibrinogen binding protein (fbe), and accumulation-associated protein (aap) genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms. The experiment was divided into 3 groups: single culture of Staphylococcus epidermidis ATCC35984 (S. epidermidis group) or Candida albicans ATCC10231 (C. albicans group), and co-culture of two strains (mixed group) to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2, 4, 6, 8, 12, 24, 48, and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy (SEM) was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of icaA, fbe, and aap genes were analyzed by real-time fluorescent quantitative PCR. Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group, and there was significant difference between 2 groups at the other time (P 0.05). In C. albicans group, the biofilm started to grow at 12 hours of cultivation, but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points (P 0.05) except at 12 hours (P 0.05); the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours (P biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe, icaA, and aap genes in mixed group increased 1.93, 1.52, and 1.46 times respectively at 72 hours compared with the S. epidermidis group (P biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans, which is related to

  9. 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 distribution of PFOS and PFOA in plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Development of green betaine-based deep eutectic solvent aqueous two-phase system for the extraction of protein.

    Science.gov (United States)

    Li, Na; Wang, Yuzhi; Xu, Kaijia; Huang, Yanhua; Wen, Qian; Ding, Xueqin

    2016-05-15

    Six kinds of new type of green betaine-based deep eutectic solvents (DESs) have been synthesized. Deep eutectic solvent aqueous two-phase systems (DES-ATPS) were established and successfully applied in the extraction of protein. Betaine-urea (Be-U) was selected as the suitable extractant. Single factor experiments were carried out to determine the optimum conditions of the extraction process, such as the salt concentration, the mass of DES, the separation time, the amount of protein, the temperature and the pH value. The extraction efficiency could achieve to 99.82% under the optimum conditions. Mixed sample and practical sample analysis were discussed. The back extraction experiment was implemented and the back extraction efficiency could reach to 32.66%. The precision experiment, repeatability experiment and stability experiment were investigated. UV-vis, FT-IR and circular dichroism (CD) spectra confirmed that the conformation of protein was not changed during the process of extraction. The mechanisms of extraction were researched by dynamic light scattering (DLS), the measurement of the conductivity and transmission electron microscopy (TEM). DES-protein aggregates and embraces phenomenon play considerable roles in the separation process. All of these results indicated that betaine-based DES-ATPS may provide a potential substitute new method for the separation of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

    Science.gov (United States)

    Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

    2004-10-01

    We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

  12. Proteomics of differential extraction fractions enriched for chromatin-binding proteins from colon adenoma and carcinoma tissues

    DEFF Research Database (Denmark)

    Knol, Jaco C; de Wit, Meike; Albrethsen, Jakob

    2014-01-01

    BACKGROUND: Altered nuclear and genomic structure and function are hallmarks of cancer cells. Research into nuclear proteins in human tissues could uncover novel molecular processes in cancer. Here, we examine biochemical tissue fractions containing chromatin-binding (CB) proteins in the context...... of colorectal cancer (CRC) progression. METHODS: CB protein-containing fractions were biochemically extracted from human colorectal tissues, including carcinomas with chromosomal instability (CIN), carcinomas with microsatellite instability (MIN), and adenomas. The CB proteins were subjected to label-free LC...

  13. PIMiner: A web tool for extraction of protein interactions from biomedical literature

    KAUST Repository

    Chowdhary, Rajesh

    2013-01-01

    Information on Protein Interactions (PIs) is valuable for biomedical research, but often lies buried in the scientific literature and cannot be readily retrieved. While much progress has been made over the years in extracting PIs from the literature using computational methods, there is a lack of free, public, user-friendly tools for the discovery of PIs. We developed an online tool for the extraction of PI relationships from PubMed-abstracts, which we name PIMiner. Protein pairs and the words that describe their interactions are reported by PIMiner so that new interactions can be easily detected within text. The interaction likelihood levels are reported too. The option to extract only specific types of interactions is also provided. The PIMiner server can be accessed through a web browser or remotely through a client\\'s command line. PIMiner can process 50,000 PubMed abstracts in approximately 7 min and thus appears suitable for large-scale processing of biological/biomedical literature. Copyright © 2013 Inderscience Enterprises Ltd.

  14. Effect of burn and first-pass splanchnic leucine extraction on protein kinetics in rats

    International Nuclear Information System (INIS)

    Karlstad, M.D.; DeMichele, S.J.; Istfan, N.; Blackburn, G.L.; Bistrian, B.R.

    1988-01-01

    The effects of burn and first-pass splanchnic leucine extraction (FPE) on protein kinetics and energy expenditure were assessed by measuring O 2 consumption, CO 2 production, nitrogen balance, leucine kinetics, and tissue fractional protein synthetic rates (FSR-%/day) in enterally fed rats. Anesthetized male rats (200 g) were scalded on their dorsum with boiling water (25-30% body surface area) and enterally fed isovolemic diets that provided 60 kcal/day and 2.4 g of amino acids/day for 3 days. Controls were not burned. An intravenous or intragastric infusion of L-[1- 14 C]leucine was used to assess protein kinetics on day 3. FPE was taken as the ratio of intragastric to intravenous plasma leucine specific activity. There was a 69% reduction in cumulative nitrogen balance (P less than 0.001) and a 17-19% increase in leucine oxidation (P less than 0.05) and total energy expenditure (P less than 0.01) in burned rats. A 15% decrease in plasma leucine clearance (P less than 0.05) was accompanied by a 20% increase in plasma [leucine] (P less than 0.01) in burned rats. Burn decreased rectus muscle FSR from 5.0 +/- 0.4 to 3.5 +/- 0.5 (P less than 0.05) and increased liver FSR from 19.0 +/- 0.5 to 39.2 +/- 3.4 (P less than 0.01). First pass extraction of dietary leucine by the splanchnic bed was 8% in controls and 26% in burned rats. Leucine kinetics corrected for FPE showed increased protein degradation with burn that was not evident without FPE correction. This hypermetabolic burn model can be useful in the design of enteral diets that optimize rates of protein synthesis and degradation

  15. Building a protein name dictionary from full text: a machine learning term extraction approach

    Directory of Open Access Journals (Sweden)

    Campagne Fabien

    2005-04-01

    Full Text Available Abstract Background The majority of information in the biological literature resides in full text articles, instead of abstracts. Yet, abstracts remain the focus of many publicly available literature data mining tools. Most literature mining tools rely on pre-existing lexicons of biological names, often extracted from curated gene or protein databases. This is a limitation, because such databases have low coverage of the many name variants which are used to refer to biological entities in the literature. Results We present an approach to recognize named entities in full text. The approach collects high frequency terms in an article, and uses support vector machines (SVM to identify biological entity names. It is also computationally efficient and robust to noise commonly found in full text material. We use the method to create a protein name dictionary from a set of 80,528 full text articles. Only 8.3% of the names in this dictionary match SwissProt description lines. We assess the quality of the dictionary by studying its protein name recognition performance in full text. Conclusion This dictionary term lookup method compares favourably to other published methods, supporting the significance of our direct extraction approach. The method is strong in recognizing name variants not found in SwissProt.

  16. Use of non-conventional cell disruption method for extraction of proteins from black yeasts

    Directory of Open Access Journals (Sweden)

    Maja eLeitgeb

    2016-04-01

    Full Text Available The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2 by varying pressure at fixed temperature (35 °C. The black yeasts cell walls were disrupted and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV-Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2 treated cells. The advantages of the proposed method are in a simple use which is also possible for heat sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions.

  17. Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

    Science.gov (United States)

    Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

    2016-01-01

    The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527

  18. Evaluation and comparison of four protein extraction protocols for mono- and two-dimensional electrophoresis in Mytilus galloprovincialis

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2015-09-01

    Full Text Available In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i TRIzol reagent; ii Lysis buffer; iii phenylmethanesulfonyl fluoride; iv trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis.

  19. Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy

    NARCIS (Netherlands)

    van der Lelij, A.; Rothova, A.; Stilma, J. S.; Hoekzema, R.; Kijlstra, A.

    1990-01-01

    Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from

  20. Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.

    Science.gov (United States)

    Kleijkers, Sander H M; van Montfoort, Aafke P A; Bekers, Otto; Coonen, Edith; Derhaag, Josien G; Evers, Johannes L H; Dumoulin, John C M

    2016-06-01

    Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation? Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C. Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice. Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements. Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days. All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet. In addition to the unstable glutamine and the protein supplements, other free amino acids might contribute to the ammonium buildup. We did not investigate the deterioration of other components in the media. Break-down of

  1. Podophyllum hexandrum (Himalayan mayapple) extract provides radioprotection by modulating the expression of proteins associated with apoptosis.

    Science.gov (United States)

    Kumar, Raj; Singh, Pankaj Kumar; Sharma, Ashok; Prasad, Jagdish; Sagar, Ravinder; Singh, Surender; Arora, Rajesh; Sharma, Rakesh Kumar

    2005-08-01

    Podophyllum hexandrum Royale (Himalayan mayapple), a high-altitude Himalayan plant, has been shown to provide over 80% whole-body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen-activated protein kinase-activated protein) kinase-2 activation along with HSF-1 (heat-shock transcription factor-1), leading to up-regulation of HSP-70 (heat-shock protein-70) as compared with sham-irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis-inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham-irradiated group. Inhibition in the translocation of free NF-kappaB (nuclear factor kappaB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation-treated mice. P. hexandrum pre-treatment (2 h before irradiation) resulted in inhibition of NF-kappaB translocation, and the expression of tumour suppressor protein p53 was observed to be down-regulated as compared with sham-irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl-2 (B-cell chronic lymphocytic lymphoma 2), Ras-GAP (Ras-GTPase-activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum-pretreated irradiated mice as compared with sham-irradiated controls. Caspase 3 activation resulted PARP [poly(ADP-ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/-irradiation) as compared with the mice treated with radiation (+/-heat shock). The present study thus clearly

  2. Proteomic Characterization of Differential Abundant Proteins Accumulated between Lower and Upper Epidermises of Fleshy Scales in Onion (Allium cepa L. Bulbs.

    Directory of Open Access Journals (Sweden)

    Si Wu

    Full Text Available The onion (Allium cepa L. is widely planted worldwide as a valuable vegetable crop. The scales of an onion bulb are a modified type of leaf. The one-layer-cell epidermis of onion scales is commonly used as a model experimental material in botany and molecular biology. The lower epidermis (LE and upper epidermis (UE of onion scales display obvious differences in microscopic structure, cell differentiation and pigment synthesis; however, associated proteomic differences are unclear. LE and UE can be easily sampled as single-layer-cell tissues for comparative proteomic analysis. In this study, a proteomic approach based on 2-DE and mass spectrometry (MS was applied to compare LE and UE of fleshy scales from yellow and red onions. We identified 47 differential abundant protein spots (representing 31 unique proteins between LE and UE in red and yellow onions. These proteins are mainly involved in pigment synthesis, stress response, and cell division. Particularly, the differentially accumulated chalcone-flavanone isomerase and flavone O-methyltransferase 1-like in LE may result in the differences in the onion scale color between red and yellow onions. Moreover, stress-related proteins abundantly accumulated in both LE and UE. In addition, the differential accumulation of UDP-arabinopyranose mutase 1-like protein and β-1,3-glucanase in the LE may be related to the different cell sizes between LE and UE of the two types of onion. The data derived from this study provides new insight into the differences in differentiation and developmental processes between onion epidermises. This study may also make a contribution to onion breeding, such as improving resistances and changing colors.

  3. The influence of protein free calf blood extract eye gel on dry eye after pterygium surgery

    Directory of Open Access Journals (Sweden)

    Cai-Ni Ji

    2013-07-01

    Full Text Available AIM: To investigate the influence of protein free calf blood extract eye gel on dry eye after pterygium surgery. METHODS: Thirty six patients(40 eyeswith primary nasal pterygium were enrolled in this study, which were divided into study group and control group randomly, with 20 eyes in each group. All patients received pterygium excision and limbal stem cell autograft surgery and tobramicin dexamethasone eye drops after surgery. Patients of the study group received protein free calf blood extract eye gel while those of the control group received 0.1% sodium hyaluronate eye drops furthermore. Ocular surface disease index(OSDIquestionnaire, tear film break-up time(BUTand Schirmer's Ⅰ test Ⅰ(SⅠtwere carried before and 3 months after surgery to evaluate the dry eye degree of the patients. RESULTS: There was no statistical difference between the age, gender and size of the pterygium of the study and control groups preoperatively. There was no statistical difference between the OSDI(2.33±1.02 vs 2.32±0.93, BUT(8.80±2.48 vs 8.35±2.28seconds and SⅠt(4.30±2.30 vs 4.40±2.44of the two groups preoperatively. There was statistical difference between the OSDI(1.45±0.47 vs 1.81±0.60, BUT(11.20±2.07 vs 9.50±2.40seconds and SⅠt(8.35±3.13 vs 6.35±2.18of the two groups 3 months postoperatively, which was also different from that of the preoperative data correspondingly. CONCLUSION: Protein free calf blood extract eye gel could reduce the dry eye after pterygium surgery.

  4. EXTRACT

    DEFF Research Database (Denmark)

    Pafilis, Evangelos; Buttigieg, Pier Luigi; Ferrell, Barbra

    2016-01-01

    The microbial and molecular ecology research communities have made substantial progress on developing standards for annotating samples with environment metadata. However, sample manual annotation is a highly labor intensive process and requires familiarity with the terminologies used. We have the...... and text-mining-assisted curation revealed that EXTRACT speeds up annotation by 15-25% and helps curators to detect terms that would otherwise have been missed.Database URL: https://extract.hcmr.gr/......., organism, tissue and disease terms. The evaluators in the BioCreative V Interactive Annotation Task found the system to be intuitive, useful, well documented and sufficiently accurate to be helpful in spotting relevant text passages and extracting organism and environment terms. Comparison of fully manual...

  5. Extraction of Protein Interaction Data: A Comparative Analysis of Methods in Use

    Directory of Open Access Journals (Sweden)

    Jose Hena

    2007-01-01

    Full Text Available Several natural language processing tools, both commercial and freely available, are used to extract protein interactions from publications. Methods used by these tools include pattern matching to dynamic programming with individual recall and precision rates. A methodical survey of these tools, keeping in mind the minimum interaction information a researcher would need, in comparison to manual analysis has not been carried out. We compared data generated using some of the selected NLP tools with manually curated protein interaction data (PathArt and IMaps to comparatively determine the recall and precision rate. The rates were found to be lower than the published scores when a normalized definition for interaction is considered. Each data point captured wrongly or not picked up by the tool was analyzed. Our evaluation brings forth critical failures of NLP tools and provides pointers for the development of an ideal NLP tool.

  6. Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates

    Science.gov (United States)

    Martin, Nicholas J.; Griffiths, Rian L.; Edwards, Rebecca L.; Cooper, Helen J.

    2015-08-01

    Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2 4H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The `contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

  7. Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

    International Nuclear Information System (INIS)

    Tao Yongguang; Song Xing; Deng Xiyun; Xie Daxin; Lee, Leo M.; Liu Yiping; Li Wei; Li Lili; Deng Lin; Wu Qiao; Gong Jianping; Cao Ya

    2005-01-01

    Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma

  8. Proteomic analysis of africanized bee venom: a comparison of protein extraction methods

    Directory of Open Access Journals (Sweden)

    Yessica Pineda Guerra

    2016-09-01

    Full Text Available The Africanised bee is the most common type of bee in Colombia, and therapeutic properties for different diseases have been attributed to its venom, without much scientific support. A literature search of reports on the proteomic analysis of honeybee venom yielded four different methods for extracting proteins from bee venom. The first method consists in resuspending the venom in 7 M Urea, followed by precipitation with acetone and finally resuspending the pellet in 7 M Urea and 4 % CHAPS. For the second method, the venom is resuspended in lysis buffer, precipitated with trichloroacetic acid, and then resuspended in 7 M Urea and 4 % CHAPS. The third method is similar to the previous one, except that the precipitation step is performed with acetone instead of trichloroacetic acid. Finally, the fourth method is to resuspend the venom in distilled water, precipitate with acetone and resuspend in 7 M Urea and 4 % CHAPS. This work focused on comparing the performance of these four extraction methods, in order to determine the method with the best results in terms of concentration and integrity of the proteins obtained. Of the four methods evaluated, the best results in terms of protein concentration and yield were obtained by resuspending the bee venom in lysis buffer followed by precipitation with acetone (method 3, and by resuspending in distilled water followed by precipitation with acetone (method 4. Of these, the method that maintained protein integrity and yielded the best proteomic profile was that in which the bee venom was resuspended in lysis buffer followed by precipitation with acetone (method 3.

  9. Identification of Protein Pupylation Sites Using Bi-Profile Bayes Feature Extraction and Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Xiaowei Zhao

    2013-01-01

    Full Text Available Pupylation, one of the most important posttranslational modifications of proteins, typically takes place when prokaryotic ubiquitin-like protein (Pup is attached to specific lysine residues on a target protein. Identification of pupylation substrates and their corresponding sites will facilitate the understanding of the molecular mechanism of pupylation. Comparing with the labor-intensive and time-consuming experiment approaches, computational prediction of pupylation sites is much desirable for their convenience and fast speed. In this study, a new bioinformatics tool named EnsemblePup was developed that used an ensemble of support vector machine classifiers to predict pupylation sites. The highlight of EnsemblePup was to utilize the Bi-profile Bayes feature extraction as the encoding scheme. The performance of EnsemblePup was measured with a sensitivity of 79.49%, a specificity of 82.35%, an accuracy of 85.43%, and a Matthews correlation coefficient of 0.617 using the 5-fold cross validation on the training dataset. When compared with other existing methods on a benchmark dataset, the EnsemblePup provided better predictive performance, with a sensitivity of 80.00%, a specificity of 83.33%, an accuracy of 82.00%, and a Matthews correlation coefficient of 0.629. The experimental results suggested that EnsemblePup presented here might be useful to identify and annotate potential pupylation sites in proteins of interest. A web server for predicting pupylation sites was developed.

  10. Criteria to Extract High-Quality Protein Data Bank Subsets for Structure Users.

    Science.gov (United States)

    Carugo, Oliviero; Djinović-Carugo, Kristina

    2016-01-01

    It is often necessary to build subsets of the Protein Data Bank to extract structural trends and average values. For this purpose it is mandatory that the subsets are non-redundant and of high quality. The first problem can be solved relatively easily at the sequence level or at the structural level. The second, on the contrary, needs special attention. It is not sufficient, in fact, to consider the crystallographic resolution and other feature must be taken into account: the absence of strings of residues from the electron density maps and from the files deposited in the Protein Data Bank; the B-factor values; the appropriate validation of the structural models; the quality of the electron density maps, which is not uniform; and the temperature of the diffraction experiments. More stringent criteria produce smaller subsets, which can be enlarged with more tolerant selection criteria. The incessant growth of the Protein Data Bank and especially of the number of high-resolution structures is allowing the use of more stringent selection criteria, with a consequent improvement of the quality of the subsets of the Protein Data Bank.

  11. Identification and characterization of finger millet OPAQUE2 transcription factor gene under different nitrogen inputs for understanding their role during accumulation of prolamin seed storage protein.

    Science.gov (United States)

    Gaur, Vikram Singh; Kumar, Lallan; Gupta, Supriya; Jaiswal, J P; Pandey, Dinesh; Kumar, Anil

    2018-03-01

    In this study, we report the isolation and characterization of the mRNA encoding OPAQUE2 (O2) like TF of finger millet (FM) ( Eleusine coracana) ( EcO2 ). Full-length EcO2 mRNA was isolated using conserved primers designed by aligning O2 mRNAs of different cereals followed by 3' and 5' RACE (Rapid Amplification of cDNA Ends). The assembled full-length EcO2 mRNA was found to contain an ORF of 1248-nt coding the 416 amino acids O2 protein. Domain analysis revealed the presence of the BLZ and bZIP-C domains which is a characteristic feature of O2 proteins. Phylogenetic analysis of EcO2 protein with other bZIP proteins identified using finger millet transcriptome data and O2 proteins of other cereals showed that EcO2 shared high sequence similarity with barley BLZ1 protein. Transcripts of EcO2 were detected in root, stem, leaves, and seed development stages. Furthermore, to investigate nitrogen responsiveness and the role of EcO2 in regulating seed storage protein gene expression, the expression profiles of EcO2 along with an α-prolamin gene were studied during the seed development stages of two FM genotypes (GE-3885 and GE-1437) differing in grain protein content (13.8 and 6.2%, respectively) grown under increasing nitrogen inputs. Compared to GE-1437, the EcO2 was relatively highly expressed during the S2 stage of seed development which further increased as nitrogen input was increased. The Ecα - prolamin gene was strongly induced in the high protein genotype (GE-3885) at all nitrogen inputs. These results indicate the presence of nitrogen responsiveness regulatory elements which might play an important role in accumulating protein in FM genotypes through modulating EcO2 expression by sensing plant nitrogen status.

  12. High Temperature During Rice Grain Filling Enhances Aspartate Metabolism in Grains and Results in Accumulation of Aspartate-Family Amino Acids and Protein Components

    Directory of Open Access Journals (Sweden)

    Cheng-gang LIANG

    2013-09-01

    Full Text Available Global warming causes the exacerbation of rice growing environment, which seriously affects rice growth and reproduction, and finally results in the decrease of rice yield and quality. We investigated the activities of aspartate metabolism enzymes in grains, and the contents of Aspartate-family amino acids and protein components to further understand the effects of high temperature (HT on rice nutritional quality during rice grain filling. Under HT, the average activities of aspartate aminotransferase (AAT and aspartokinase (AK in grains significantly increased, the amino acid contents of aspartate (Asp, lysine (Lys, threonine (Thr, methionine (Met and isoleucine (Ile and the protein contents of albumin, globulin, prolamin and glutelin also significantly increased. The results indicated that HT enhanced Asp metabolism during rice grain filling and the enhancement of Asp metabolism might play an important role in the increase of Asp-family amino acids and protein components in grains. In case of the partial appraisal of the change of Asp-family amino acids and protein components under HT, we introduced eight indicators (amino acid or protein content, ratio of amino acid or protein, amino acid or protein content per grain and amino acid or protein content per panicle to estimate the effects of HT. It is suggested that HT during rice grain filling was benefit for the accumulation of Asp-family amino acids and protein components. Combined with the improvement of Asp-family amino acid ratio in grains under HT, it is suggested that HT during grain filling may improve the rice nutritional quality. However, the yields of parts of Asp-family amino acids and protein components were decreased under HT during rice grain filling.

  13. An extract of Urtica dioica L. mitigates obesity induced insulin resistance in mice skeletal muscle via protein phosphatase 2A (PP2A).

    Science.gov (United States)

    Obanda, Diana N; Ribnicky, David; Yu, Yongmei; Stephens, Jacqueline; Cefalu, William T

    2016-02-26

    The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation.

  14. AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xia Luo

    2015-01-01

    Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

  15. Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

    Directory of Open Access Journals (Sweden)

    Arumugam Muthu

    2016-11-01

    Full Text Available Abiotic stress in oleaginous microalgae enhances lipid accumulation and is stored in a specialised organelle called lipid droplets (LDs. Both the LDs and body are enriched with major lipid droplet protein (MLDP. It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of S. quadricauda under the salt stress of 10mM concentration is about 0.174μ and in control, the SGR is 0.241μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17. The dry biomass content also decreased drastically at 50mM salt-treated cells (129mg/L compared to control (236mg/L on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

  16. Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

    International Nuclear Information System (INIS)

    Javee, Anand; Sulochana, Sujitha Balakrishnan; Pallissery, Steffi James; Arumugam, Muthu

    2016-01-01

    Abiotic stress in oleaginous microalgae enhances lipid accumulation, which is stored in a specialized organelle called lipid droplets (LDs). Both the LDs or lipid body are enriched with major lipid droplet protein (MLDP). It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of Scenedesmus quadricauda under the salt stress of 10mM concentration is about 0.174 μ and in control, the SGR is 0.241 μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17). The dry biomass content also decreased drastically at 50mM salt-treated cells (129 mg/L) compared to control (236 mg/L) on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

  17. Major Lipid Body Protein: A Conserved Structural Component of Lipid Body Accumulated during Abiotic Stress in S. quadricauda CASA-CC202

    Energy Technology Data Exchange (ETDEWEB)

    Javee, Anand [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Sulochana, Sujitha Balakrishnan [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India); Pallissery, Steffi James [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Arumugam, Muthu, E-mail: arumugam@niist.res.in [Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India)

    2016-11-23

    Abiotic stress in oleaginous microalgae enhances lipid accumulation, which is stored in a specialized organelle called lipid droplets (LDs). Both the LDs or lipid body are enriched with major lipid droplet protein (MLDP). It serves as a major structural component and also plays a key role in recruiting other proteins and enzymes involved in lipid body maturation. In the present study, the presence of MLDP was detected in two abiotic stress condition namely nitrogen starvation and salt stress condition. Previous research reveals that nitrogen starvation enhances lipid accumulation. Therefore, the effect of salt on growth, biomass yield, and fatty acid profile is studied in detail. The specific growth rate of Scenedesmus quadricauda under the salt stress of 10mM concentration is about 0.174 μ and in control, the SGR is 0.241 μ. An increase in the doubling time of the cells shows that the rate of cell division decreases during salt stress (2.87–5.17). The dry biomass content also decreased drastically at 50mM salt-treated cells (129 mg/L) compared to control (236 mg/L) on the day 20. The analysis of fatty acid composition also revealed that there is a 20% decrease in the saturated fatty acid level and 19.9% increment in monounsaturated fatty acid level, which makes salt-mediated lipid accumulation as a suitable biodiesel precursor.

  18. Nickel accumulation and storage in bradyrhizobium japonicum

    International Nuclear Information System (INIS)

    Maier, R.J.; Pihl, T.D.; Stults, L.; Sray, W.

    1990-01-01

    Hydrogenase-depressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl 2 , and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown with Ni and the derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63 Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunuprecipitation of 63 Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms

  19. Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

    Science.gov (United States)

    Vaganan, M Mayil; Sarumathi, S; Nandakumar, A; Ravi, I; Mustaffa, M M

    2015-02-01

    Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

  20. Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    DEFF Research Database (Denmark)

    Callesen, Anne K; Mohammed, Shabaz; Bunkenborg, Jakob

    2005-01-01

    for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI...

  1. Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

    NARCIS (Netherlands)

    Ruiz, Geraldine Avila; Opazo-Navarrete, Mauricio; Meurs, Marlon; Minor, Marcel; Sala, Guido; Boekel, van Tiny; Stieger, Markus; Janssen, Anja E.M.

    2016-01-01

    The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60,

  2. Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens.

    Science.gov (United States)

    Satoh, Rie; Teshima, Reiko; Kitta, Kazumi; Lang, Gang-Hua; Schegg, Kathleen; Blumenthal, Kenneth; Hicks, Leslie; Labory-Carcenac, Bénédicte; Rouquié, David; Herman, Rod A; Herouet-Guicheney, Corinne; Ladics, Gregory S; McClain, Scott; Poulsen, Lars K; Privalle, Laura; Ward, Jason M; Doerrer, Nancy; Rascle, Jean-Baptiste

    2016-07-11

    In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optimal rice extraction and detection methods that would allow a sensitive and reproducible measure of several classes of known rice allergens. In a three-laboratory ring-trial experiment, several protein extraction methods were first compared and analyzed by 1D multiplexed SDS-PAGE. In a second phase, an inter-laboratory validation of 2D-DIGE analysis was conducted in five independent laboratories, focusing on three rice allergens (52 kDa globulin, 33 kDa glyoxalase I, and 14-16 kDa α-amylase/trypsin inhibitor family members). The results of the present study indicate that a combination of 1D multiplexed SDS-PAGE and 2D-DIGE methods would be recommended to quantify the various rice allergens.

  3. Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

    Science.gov (United States)

    Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

    2014-07-04

    Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

  4. Screening of immunomodulatory activity of total and protein extracts of some Moroccan medicinal plants.

    Science.gov (United States)

    Daoudi, Abdeljlil; Aarab, Lotfi; Abdel-Sattar, Essam

    2013-04-01

    Herbal and traditional medicines are being widely used in practice in many countries for their benefits of treating different ailments. A large number of plants in Morocco were used in folk medicine to treat immune-related disorders. The objective of this study is to evaluate the immunomodulatory activity of protein extracts (PEs) of 14 Moroccan medicinal plants. This activity was tested on the proliferation of immune cells. The prepared total and PEs of the plant samples were tested using MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay on the splenocytes with or without stimulation by concanavalin-A (Con-A), a mitogenic agent used as positive control. The results of this study indicated different activity spectra. Three groups of activities were observed. The first group represented by Citrullus colocynthis, Urtica dioica, Elettaria cardamomum, Capparis spinosa and Piper cubeba showed a significant immunosuppressive activity. The second group that showed a significant immunostimulatory activity was represented by Aristolochia longa, Datura stramonium, Marrubium vulgare, Sinapis nigra, Delphynium staphysagria, Lepidium sativum, Ammi visnaga and Tetraclinis articulata. The rest of the plant extracts did not alter the proliferation induced by Con-A. This result was more important for the PE than for the total extract. In conclusion, this study revealed an interesting immunomodulating action of certain PEs, which could explain their traditional use. The results of this study may also have implications in therapeutic treatment of infections, such as prophylactic and adjuvant with cancer chemotherapy.

  5. Enhanced accumulation of Kir4.1 protein, but not mRNA, in a murine model of cuprizone-induced demyelination.

    Science.gov (United States)

    Nakajima, Mitsunari; Kawamura, Takuya; Tokui, Ryuji; Furuta, Kohei; Sugino, Mami; Nakanishi, Masayuki; Okuyama, Satoshi; Furukawa, Yoshiko

    2013-11-06

    Two channel proteins, inwardly rectifying potassium channel 4.1 (Kir4.1) and water channel aquaporin-4 (AQP4), were recently identified as targets of an autoantibody response in patients with multiple sclerosis and neuromyelitis optica, respectively. In the present study, we examined the expression patterns of Kir4.1 and AQP4 in a mouse model of demyelination induced by cuprizone, a copper chelator. Demyelination was confirmed by immunohistochemistry using an anti-proteolipid protein antibody in various brain regions, including the corpus callosum, of cuprizone-fed mice. Activation of microglial and astroglial cells was also confirmed by immunohistochemistry, using an anti-ionized calcium binding adapter molecule and a glial fibrillary acidic protein antibody. Western blot analysis revealed the induction of Kir4.1 protein, but not AQP4, in the cortex of cuprizone-fed mice. Immunohistochemical analysis confirmed the Kir4.1 protein induction in microvessels of the cerebral cortex. Real-time polymerase chain reaction analysis revealed that mRNA levels of Kir4.1 and AQP4 in the cortex did not change during cuprizone administration. These findings suggest that enhanced accumulation of Kir4.1 protein in the brain with an inflammatory condition facilitates the autoantibody formation against Kir4.1 in patients with multiple sclerosis. © 2013 Published by Elsevier B.V.

  6. Proteomic analysis of the maize rachis: potential roles of constitutive and induced proteins in resistance to Aspergillus flavus infection and aflatoxin accumulation.

    Science.gov (United States)

    Pechanova, Olga; Pechan, Tibor; Williams, W Paul; Luthe, Dawn S

    2011-01-01

    Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Protein content and electrophoretic profile of fat body and ovary extracts from workers of Melipona quadrifasciata anthidioides (Hymenoptera, Meliponini

    Directory of Open Access Journals (Sweden)

    Vagner T. Paes de Oliveira

    Full Text Available Workers of Melipona quadrifasciata anthidioides (Lepeletier, 1836 develop their ovaries and lay eggs, therefore the production of vitellogenin is expected. In electrophoretic profiles only fat body extracts from nurse workers and ovary extracts from newly-emerged workers show protein with molecular mass similar to vitellogenin. However, an increase in the protein content was detected in forager fat body. This increase was attributed to storage of vitellogenin or other proteins in the previous phase and not discharged into the hemolymph or to an effect of the increased titre of juvenile hormone in this phase of worker life over the fat body functioning.

  8. Enzyme-treated asparagus extract promotes expression of heat shock protein and exerts antistress effects.

    Science.gov (United States)

    Ito, Tomohiro; Maeda, Takahiro; Goto, Kazunori; Miura, Takehito; Wakame, Koji; Nishioka, Hiroshi; Sato, Atsuya

    2014-03-01

    A novel enzyme-treated asparagus extract (ETAS) has been developed as a functional material produced from asparagus stem. Studies were conducted to determine the effect of ETAS on heat shock protein 70 (HSP70) expression and alleviation of stress. HeLa cells were treated with ETAS, and HSP70 mRNA and protein levels were measured using a reverse transcription-polymerase chain reaction (RT-PCR) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. ETAS showed significant increases in HSP70 mRNA at more than 0.125 mg/mL and the protein at more than 1.0 mg/mL. The antistress effect was evaluated in a murine sleep-deprivation model. A sleep-deprivation stress load resulted in elevation of blood corticosterone and lipid peroxide concentrations, while supplementation with ETAS at 200 and 1000 mg/kg body weight was associated with significantly reduced levels of both stress markers, which were in the normal range. The HSP70 protein expression level in mice subjected to sleep-deprivation stress and supplemented with ETAS was significantly enhanced in stomach, liver, and kidney, compared to ETAS-untreated mice. A preliminary and small-sized human study was conducted among healthy volunteers consuming up to 150 mg/d of ETAS daily for 7 d. The mRNA expression of HSP70 in peripheral leukocytes was significantly elevated at intakes of 100 or 150 mg/d, compared to their baseline levels. Since HSP70 is known to be a stress-related protein and its induction leads to cytoprotection, the present results suggest that ETAS might exert antistress effects under stressful conditions, resulting from enhancement of HSP70 expression. © 2014 Institute of Food Technologists®

  9. Accumulation of sphingolipid activator proteins (SAPs) A and D in granular osmiophilic deposits in miniature Schnauzer dogs with ceroid-lipofuscinosis.

    Science.gov (United States)

    Palmer, D N; Tyynelä, J; van Mil, H C; Westlake, V J; Jolly, R D

    1997-03-01

    The neuronal ceroid-lipofuscinoses (NCL, Batten disease) are fatal inherited neurodegenerative diseases of children characterized by retinal and brain atrophy and the accumulation of electron-dense storage bodies in cells. Mutations in different genes underlie different major forms. The infantile disease (CLN-1, McKusick 256730) is distinguished by the storage of the sphingolipid activator proteins (SAPs) A and D in distinctive granular osmiophilic deposits (GRODs). This contrasts with the other major forms, where subunit c of mitochondrial ATP synthase is stored in various multilamellar profiles. Ceroid-lipofuscinoses also occur in dogs, including a form in miniature Schnauzers with distinctive granular osmiophilic deposit-like storage bodies. Antisera to SAPs A and D reacted to these storage bodies in situ. The presence of SAP D was confirmed by Western blotting and of SAP A by protein sequencing. Neither subunit c of mitochondrial ATP synthase nor of vacuolar ATPase is stored. This suggests that there are two families of ceroid-lipofuscinoses, the subunit c-storing forms, and those in which SAPs A and D, and perhaps other proteins, accumulate. Further work is required to determine whether other forms with granular osmiophilic deposits belong to the latter class and the genetic relationships between them and the human infantile disease.

  10. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    Science.gov (United States)

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. © 2016 The Royal Entomological Society.

  11. Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets.

    Science.gov (United States)

    Iglesias, Nestor G; Mondotte, Juan A; Byk, Laura A; De Maio, Federico A; Samsa, Marcelo M; Alvarez, Cecilia; Gamarnik, Andrea V

    2015-09-01

    Dengue viruses cause the most important human viral disease transmitted by mosquitoes. In recent years, a great deal has been learned about molecular details of dengue virus genome replication; however, little is known about genome encapsidation and the functions of the viral capsid protein. During infection, dengue virus capsid progressively accumulates around lipid droplets (LDs) by an unknown mechanism. Here, we examined the process by which the viral capsid is transported from the endoplasmic reticulum (ER) membrane, where the protein is synthesized, to LDs. Using different methods of intervention, we found that the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transport to LDs, while the process is independent of both COPII components and Golgi integrity. The transport was sensitive to Brefeldin A, while a drug resistant form of GBF1 was sufficient to restore capsid subcellular distribution in infected cells. The mechanism by which LDs gain or lose proteins is still an open question. Our results support a model in which the virus uses a non-canonical function of the COPI system for capsid accumulation on LDs, providing new ideas for antiviral strategies. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Transforming growth factor β-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

    Science.gov (United States)

    McNary, Sean M; Athanasiou, Kyriacos A; Reddi, A Hari

    2014-03-01

    The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor β (TGFβ) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFβ1. A decrease in TGFβ1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFβ-induced SZP accumulation. TGFβ-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1

  13. Differential Proteome Analysis of the Preeclamptic Placenta Using Optimized Protein Extraction

    Directory of Open Access Journals (Sweden)

    Magnus Centlow

    2010-01-01

    Full Text Available The human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen. Both lipids and glycogen are known to interfere with the first step in the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE, the isoelectric focusing. In order to gain the best possible protein separation on 2D-PAGE, an optimized sample preparation protocol for placental proteins was developed. Two different buffers, urea/CHAPS and Hepes, were used for solubilization in combination with six different precipitation methods. The removal of glycogen from the samples by centrifugation was crucial for the final proteome maps. Solubilization with urea/CHAPS in combination with dichloromethane/methanol or acidified acetone proved to be the best precipitation procedures. When applied to clinical placenta samples apolipoprotein A1 was found to be accumulated in the preeclamptic placenta, where it may either have a nutritional effect or act as a modifier of signal transduction.

  14. Palatability of water-soluble extracts of protein sources and replacement of fishmeal by a selected mixture of protein sources for juvenile turbot ( Scophthalmus maximus)

    Science.gov (United States)

    Dong, Chun; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei

    2016-06-01

    Poor palatability is a limiting factor for replacing fishmeal with other protein sources in aquaculture. The water-soluble molecules with low molecular weights are the major determinants of the palatability of diets. The present study was conducted to investigate the palatability of water-soluble extracts from single protein source (single extract pellets) and the mixture of these extracts with different proportions (blended extract pellets) in juvenile turbot ( Scophthalmus maximus). Then according to the palatability of blended extract pellets, an optimal mixture proportion was selected, and a new protein source made from raw protein materials with the selected proportion was formulated to replace fishmeal. Summarily, the palatability of single extract pellets for turbot was descendent from fishmeal to pet-food grade poultry by-product meal, wheat gluten meal, soybean meal, peanut meal, meat and bone meal, and corn gluten meal. Subsequently, according to the palatability of single extract pellets, 52 kinds of blended extract pellets were designed to test their palatability. The results showed that the pellets presented remarkably different palatability, and the optimal one was diet 52 (wheat gluten meal: pet-food grade poultry by-product meal: meat and bone meal: corn gluten meal = 1:6:1:2). The highest ingestion ratio (the number of pellets ingested/the number of pellets fed) was 0.73 ± 0.03, which was observed in Diet 52. Then five isonitrogenous (52% crude protein) and isocaloric (20 kJ g-1 gross energy) diets were formulated by replacing 0 (control), 35%, 50%, 65% and 80% of fishmeal with No.52 blending proportion. After a 10-weeks feeding trial, a consistent feed intake was found among all replacement treatments. Replacement level of fishmeal up to 35% did not significantly influence final body weight, specific growth rate, feed efficiency ratio, and protein efficiency ratio of turbot. Therefore, the water-soluble extracts of protein sources play an

  15. Evaluation of sonication treatment and buffer composition on rumen bacteria protein extraction and carboxymethylcellulase activity.

    Science.gov (United States)

    Prauchner, Carlos A; Kozloski, Gilberto V; Farenzena, Roberta

    2013-05-01

    The methodological procedures for studying the fibrolytic activity of rumen bacteria are not clearly established. In this study the efficiency of sonication treatment and buffer composition (i.e. buffer varying in tonicity or pH) on the level of protein extraction from the residue of forage samples incubated in the rumen of a grazing steer and the effect of buffer composition or CaCl₂ concentration on the carboxymethylcellulase (CMCase) activity of the released protein were evaluated. The amount of protein released from the residue of incubation was higher (P buffer pH (P buffer. Both linear and quadratic effects (P buffer pH on CMCase activity were significant, with CMCase activity being maximal at pH 5.4-6.1. CMCase activity was higher (P buffer solution that includes a calcium source. When pH is not a treatment factor, the buffer pH should be between 5.5 and 6. © 2012 Society of Chemical Industry.

  16. Characterization of enzymatically extracted sunflower seed oil as well as the protein residues

    Directory of Open Access Journals (Sweden)

    Sitohy, M. Z.

    1993-12-01

    Full Text Available Sunflower seed oil was enzymatically extracted with six different enzymes: cellulase, hemicellulase, animal proteinase, acid proteinase, pectinase and pectinex under the following conditions: substrate concentration in phosphate buffer (0.5M, pH 5 30%, enzyme concentration 2% (E/S, temperature 50°C and time 3 hours. The obtained oils were analyzed for physicochemical properties and fatty acid profiles. The protein residues were analyzed for amino acid compositions. The results showed that the enzymatic extraction with cellulase or hemicellulase could maintain good oil quality of the extracted oils as their levels of linoleic and oleic acids recorded similar values to those of the control oil extracted with organic solvents. Also the level of iodine value was in the same level of control. On the other hand, the use of proteases in the enzymatic extraction of sunflower seed oil caused some reductions in the levels of the unsaturated fatty acids as well as the iodine value. The pectinases showed a similar trend to that of the proteinase with the least recovery of linoleic acid among the different oils under study. Similarly, the use of cellulases did not change the amino acid composition of the protein residue as compared to the control, in the contrary to the extraction with the proteinases which caused reduction of some amino acids from the protein residues especially lysine, leucine, iso-leucine, alanine, arginine and aspartic. In that respect the use of pectinases behaved similar to cellulases.

    Aceite de semilla de girasol fue extraído enzimáticamente con seis enzimas diferentes: celulasa, hemicelulasa, proteinasa animal, proteinase acida, pectinasa y pectinex bajo las condiciones siguientes: concentración de sustrato en tampón fosfato (0,5M, pH 5 30%, concentración enzimática 2% (E/S, temperatura 50°C y tiempo 3 horas. Los aceites obtenidos fueron analizados por sus propiedades fisicoquímicas y perfiles de ácidos grasos

  17. Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

    Science.gov (United States)

    Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

    2017-07-01

    Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels

  18. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    Science.gov (United States)

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  19. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  20. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5′-phosphate-binding protein YggS

    OpenAIRE

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-01-01

    Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/M...

  1. Magnetic graphene oxide modified with choline chloride-based deep eutectic solvent for the solid-phase extraction of protein.

    Science.gov (United States)

    Huang, Yanhua; Wang, Yuzhi; Pan, Qi; Wang, Ying; Ding, Xueqin; Xu, Kaijia; Li, Na; Wen, Qian

    2015-06-02

    Four kinds of green deep eutectic solvents (DESs) based on choline chloride (ChCl) have been synthesized and coated on the surface of magnetic graphene oxide (Fe3O4@GO) to form Fe3O4@GO-DES for the magnetic solid-phase extraction of protein. X-ray diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FTIR), field emission scanning electron microscopy (FESEM) and thermal gravimetric analysis (TGA) were employed to characterize Fe3O4@GO-DES, and the results indicated the successful preparation of Fe3O4@GO-DES. The UV-vis spectrophotometer was used to measure the concentration of protein after extraction. Single factor experiments proved that the extraction amount was influenced by the types of DESs, solution temperature, solution ionic strength, extraction time, protein concentration and the amount of Fe3O4@GO-DES. Comparison of Fe3O4@GO and Fe3O4@GO-DES was carried out by extracting bovine serum albumin, ovalbumin, bovine hemoglobin and lysozyme. The experimental results showed that the proposed Fe3O4@GO-DES performs better than Fe3O4@GO in the extraction of acidic protein. Desorption of protein was carried out by eluting the solid extractant with 0.005 mol L(-1) Na2HPO4 contained 1 mol L(-1) NaCl. The obtained elution efficiency was about 90.9%. Attributed to the convenient magnetic separation, the solid extractant could be easily recycled. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    Directory of Open Access Journals (Sweden)

    Xiang Y. Kong

    2014-03-01

    Full Text Available Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.

  3. Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd)

    NARCIS (Netherlands)

    Ruiz, Geraldine Avila; Xiao, Wukai; Boekel, van Tiny; Minor, Marcel; Stieger, Markus

    2016-01-01

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8

  4. Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins.

    Science.gov (United States)

    Gai, Qingqing; Qu, Feng; Zhang, Tao; Zhang, Yukui

    2011-07-15

    Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic ProteinsW⃞

    Science.gov (United States)

    Kubis, Sybille; Baldwin, Amy; Patel, Ramesh; Razzaq, Azam; Dupree, Paul; Lilley, Kathryn; Kurth, Joachim; Leister, Dario; Jarvis, Paul

    2003-01-01

    The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins. PMID:12897258

  6. The peculiarities of 137Cs accumulation by plants of meadow phytocenosis and content of photosynthetic pigments and protein in the plant leaves

    International Nuclear Information System (INIS)

    Gaponenko, V.; Kravchenko, V.; Matsko, V.

    1994-01-01

    In connection with the Chernobyl accident the problem of resistance of different plant systems, including photosynthesis, in conditions of increased background radiation and radioactive soil contamination was investigated. The dynamics of pigments and proteins in the leaves of meadow phytocenosis at different contamination levels of Polesski State was studied. The reverse correlation was discovered between the coefficients of radionuclide accumulation by plants and the specific radioactivity of soil. A prevailing decrease in concentration of chlorophyll 'b' as compared to chlorophyll 'a' was discovered as well as a decrease in both green pigments as compared to carotenoids at the end of vegetation. At the lowest specific 137 Cs radioactivity in fresh leaf tissues of Agropyron repens L. the content of pigments in leaves was biggest. A positive correlation between specific radioactivity of overground phytomass (Bq/kg) and protein concentration in leaves was revealed. An analogous relationship has appeared too for one year old and two year old needles of Pinus Silvestris L. (author)

  7. Accumulation of radium in ferruginous protein bodies formed in lung tissue. Association of resulting radiation hotspots with malignant mesothelioma and other malignancies

    International Nuclear Information System (INIS)

    Nakamura, Eizo; Makishima, Akio; Hagino, Kyoko; Okabe, Kazunori

    2009-01-01

    While exposure to fibers and particles has been proposed to be associated with several different lung malignancies including mesothelioma, the mechanism for the carcinogenesis is not fully understood. Along with mineralogical observation, we have analyzed forty-four major and trace elements in extracted asbestos bodies (fibers and proteins attached to them) with coexisting fiber-free ferruginous protein bodies from extirpative lungs of individuals with malignant mesothelioma. These observations together with patients' characteristics suggest that inhaled iron-rich asbestos fibers and dust particles, and excess iron deposited by continuous cigarette smoking would induce ferruginous protein body formation resulting in ferritin aggregates in lung tissue. Chemical analysis of ferruginous protein bodies extracted from lung tissues reveals anomalously high concentrations of radioactive radium, reaching millions of times higher concentration than that of seawater. Continuous and prolonged internal exposure to hotspot ionizing radiation from radium and its daughter nuclides could cause strong and frequent DNA damage in lung tissue, initiate different types of tumour cells, including malignant mesothelioma cells, and may cause cancers. (author)

  8. Membrane protein extraction and purification using styrene-maleic acid (SMA) copolymer: effect of variations in polymer structure.

    Science.gov (United States)

    Morrison, Kerrie A; Akram, Aneel; Mathews, Ashlyn; Khan, Zoeya A; Patel, Jaimin H; Zhou, Chumin; Hardy, David J; Moore-Kelly, Charles; Patel, Roshani; Odiba, Victor; Knowles, Tim J; Javed, Masood-Ul-Hassan; Chmel, Nikola P; Dafforn, Timothy R; Rothnie, Alice J

    2016-12-01

    The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  9. Protein Profiles for Muscle Development and Intramuscular Fat Accumulation at Different Post-Hatching Ages in Chickens.

    Directory of Open Access Journals (Sweden)

    Jie Liu

    Full Text Available Muscle development and growth influences the efficiency of poultry meat production, and is closely related to deposition of intramuscular fat (IMF, which is crucial in meat quality. To clarify the molecular mechanisms underlying muscle development and IMF deposition in chickens, protein expression profiles were examined in the breast muscle of Beijing-You chickens at ages 1, 56, 98 and 140 days, using isobaric tags for relative and absolute quantification (iTRAQ. Two hundred and four of 494 proteins were expressed differentially. The expression profile at day 1 differed greatly from those at day 56, 98 and 140. KEGG pathway analysis of differential protein expression from pair-wise comparisons (day 1 vs. 56; 56 vs. 98; 98 vs. 140, showed that the fatty acid degradation pathway was more active during the stage from day 1 to 56 than at other periods. This was consistent with the change in IMF content, which was highest at day 1 and declined dramatically thereafter. When muscle growth was most rapid (days 56-98, pathways involved in muscle development were dominant, including hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, tight junctions and focal adhesion. In contrast with hatchlings, the fatty acid degradation pathway was downregulated from day 98 to 140, which was consistent with the period for IMF deposition following rapid muscle growth. Changes in some key specific proteins, including fast skeletal muscle troponin T isoform, aldehyde dehydrogenase 1A1 and apolipoprotein A1, were verified by Western blotting, and could be potential biomarkers for IMF deposition in chickens. Protein-protein interaction networks showed that ribosome-related functional modules were clustered in all three stages. However, the functional module involved in the metabolic pathway was only clustered in the first stage (day 1 vs. 56. This study improves our understanding of the molecular mechanisms underlying muscle development and IMF

  10. Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes.

    Science.gov (United States)

    Ytterberg, A Jimmy; Peltier, Jean-Benoit; van Wijk, Klaas J

    2006-03-01

    Plastoglobules (PGs) are oval or tubular lipid-rich structures present in all plastid types, but their specific functions are unclear. PGs contain quinones, alpha-tocopherol, and lipids and, in chromoplasts, carotenoids as well. It is not known whether PGs contain any enzymes or regulatory proteins. Here, we determined the proteome of PGs from chloroplasts of stressed and unstressed leaves of Arabidopsis (Arabidopsis thaliana) as well as from pepper (Capsicum annuum) fruit chromoplasts using mass spectrometry. Together, this showed that the proteome of chloroplast PGs consists of seven fibrillins, providing a protein coat and preventing coalescence of the PGs, and an additional 25 proteins likely involved in metabolism of isoprenoid-derived molecules (quinines and tocochromanols), lipids, and carotenoid cleavage. Four unknown ABC1 kinases were identified, possibly involved in regulation of quinone monooxygenases. Most proteins have not been observed earlier but have predicted N-terminal chloroplast transit peptides and lack transmembrane domains, consistent with localization in the PG lipid monolayer particles. Quantitative differences in PG composition in response to high light stress and degreening were determined by differential stable-isotope labeling using formaldehyde. More than 20 proteins were identified in the PG proteome of pepper chromoplasts, including four enzymes of carotenoid biosynthesis and several homologs of proteins observed in the chloroplast PGs. Our data strongly suggest that PGs in chloroplasts form a functional metabolic link between the inner envelope and thylakoid membranes and play a role in breakdown of carotenoids and oxidative stress defense, whereas PGs in chromoplasts are also an active site for carotenoid conversions.

  11. Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes1[W

    Science.gov (United States)

    Ytterberg, A. Jimmy; Peltier, Jean-Benoit; van Wijk, Klaas J.

    2006-01-01

    Plastoglobules (PGs) are oval or tubular lipid-rich structures present in all plastid types, but their specific functions are unclear. PGs contain quinones, α-tocopherol, and lipids and, in chromoplasts, carotenoids as well. It is not known whether PGs contain any enzymes or regulatory proteins. Here, we determined the proteome of PGs from chloroplasts of stressed and unstressed leaves of Arabidopsis (Arabidopsis thaliana) as well as from pepper (Capsicum annuum) fruit chromoplasts using mass spectrometry. Together, this showed that the proteome of chloroplast PGs consists of seven fibrillins, providing a protein coat and preventing coalescence of the PGs, and an additional 25 proteins likely involved in metabolism of isoprenoid-derived molecules (quinines and tocochromanols), lipids, and carotenoid cleavage. Four unknown ABC1 kinases were identified, possibly involved in regulation of quinone monooxygenases. Most proteins have not been observed earlier but have predicted N-terminal chloroplast transit peptides and lack transmembrane domains, consistent with localization in the PG lipid monolayer particles. Quantitative differences in PG composition in response to high light stress and degreening were determined by differential stable-isotope labeling using formaldehyde. More than 20 proteins were identified in the PG proteome of pepper chromoplasts, including four enzymes of carotenoid biosynthesis and several homologs of proteins observed in the chloroplast PGs. Our data strongly suggest that PGs in chloroplasts form a functional metabolic link between the inner envelope and thylakoid membranes and play a role in breakdown of carotenoids and oxidative stress defense, whereas PGs in chromoplasts are also an active site for carotenoid conversions. PMID:16461379

  12. Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

    Science.gov (United States)

    Mora-Santos, Mar; Limón-Mortés, M Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

    2011-08-26

    PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.

  13. Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ishii Marina

    2002-04-01

    Full Text Available Abstract Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside, express the green fluorescent protein (gfpuv during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1 of transformed (pGFP Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm were sonicated in successive intervals of sonication (25 vibrations/pulse to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations. The intracellular permeate with gfpuv in extraction buffer (TE solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF was subjected to the three-phase partitioning (TPP method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0. Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA, after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP

  14. Flour sodium dodecyl sulfate (SDS)-extractable protein level as a cookie flour quality indicator.

    Science.gov (United States)

    Pareyt, Bram; Bruneel, Charlotte; Brijs, Kristof; Goesaert, Hans; Delcour, Jan A

    2010-01-13

    Flour characteristics of laboratory-milled flour fractions of two wheat cultivars were related to their cookie-baking performance. Cultivar (cv.) Albatros wheat milling yielded fractions with lower damaged starch (DS) and arabinoxylan levels and higher sodium dodecyl sulfate-extractable protein (SDSEP) levels than did cv. Meunier wheat milling. During baking, cv. Albatros flour doughs spread faster and set later than their cv. Meunier counterparts and, hence, resulted in larger cookie diameters. DS levels negatively affected spread rate during both cv. Albatros (R2=0.68) and cv. Meunier (R2=0.51) cookie baking. SDSEP levels also influenced cookie quality. The use of flour heat-treated to reduce its SDSEP levels to different degrees led to reduction of the set time (R2=0.90). It was deduced that larger gluten polymer sizes limit dough spread time during baking and that, apart from DS level, the SDSEP level is an indicator for cookie flour quality.

  15. Interactions Between Flavonoid-Rich Extracts and Sodium Caseinate Modulate Protein Functionality and Flavonoid Bioaccessibility in Model Food Systems.

    Science.gov (United States)

    Elegbede, Jennifer L; Li, Min; Jones, Owen G; Campanella, Osvaldo H; Ferruzzi, Mario G

    2018-05-01

    With growing interest in formulating new food products with added protein and flavonoid-rich ingredients for health benefits, direct interactions between these ingredient classes becomes critical in so much as they may impact protein functionality, product quality, and flavonoids bioavailability. In this study, sodium caseinate (SCN)-based model products (foams and emulsions) were formulated with grape seed extract (GSE, rich in galloylated flavonoids) and green tea extract (GTE, rich in nongalloylated flavonoids), respectively, to assess changes in functional properties of SCN and impacts on flavonoid bioaccessibility. Experiments with pure flavonoids suggested that galloylated flavonoids reduced air-water interfacial tension of 0.01% SCN dispersions more significantly than nongalloylated flavonoids at high concentrations (>50 μg/mL). This observation was supported by changes in stability of 5% SCN foam, which showed that foam stability was increased at high levels of GSE (≥50 μg/mL, P < 0.05) but was not affected by GTE. However, flavonoid extracts had modest effects on SCN emulsion. In addition, galloylated flavonoids had higher bioaccessibility in both SCN foam and emulsion. These results suggest that SCN-flavonoid binding interactions can modulate protein functionality leading to difference in performance and flavonoid bioaccessibility of protein-based products. As information on the beneficial health effects of flavonoids expands, it is likely that usage of these ingredients in consumer foods will increase. However, the necessary levels to provide such benefits may exceed those that begin to impact functionality of the macronutrients such as proteins. Flavonoid inclusion within protein matrices may modulate protein functionality in a food system and modify critical consumer traits or delivery of these beneficial plant-derived components. The product matrices utilized in this study offer relevant model systems to evaluate how fortification with flavonoid

  16. Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of IκBα and nuclear accumulation of NF-κB

    International Nuclear Information System (INIS)

    Maier, Jana V.; Volz, Yvonne; Berger, Caroline; Schneider, Sandra; Cato, Andrew C.B.

    2010-01-01

    Research highlights: →Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-κB. →Bag-1 depletion attenuates phosphorylation and degradation of IκBα and nuclear accumulation of NF-κB p65 and p50. →Bag-1 interacts with IκBα and partially restores IκBα and NF-κB activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulate the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-κB. Furthermore phosphorylation and degradation of the inhibitor protein IκBα and nuclear accumulation of p65 and p50 NF-κB proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored IκBα and NF-κB activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.

  17. The protective ability of Camellia meal extract on the silk protein

    Science.gov (United States)

    Weng, JZ; Cai, C.; Zhang, DY; Dai, BK

    2018-02-01

    With the enhancement of living standards, people pay more and more attention to the health. The edible oil become more and more popular, but also produced a large amount of Camellia meal which can not fully put into utilization. In this study, the extracting liquid of Camellia meal was used on the process of silk degumming. Firstly, tussah silk was treated by degumming in the Na2CO3 solution, and the preliminary condition of tussah silk degumming was obtained by orthogonal experiment: the concentration Na2CO3 was 0.1%, the degumming time was 1 hour, and the ratio of silk/water was 40:1. Then the extract of Camellia meal (GCJSY) was added before the bleaching process of tussah silk to investigate the protective ability of GCJSY on the silk protein basry on the residual ratio of the silk. While the concentration of GYJSY was 0.08%, the residual ratio of silk after degumming in the Na2CO3 solution and bleaching in the 2% H2O2 solution was 87.2%.

  18. Heat-induced accumulation of protein synthesis elongation factor 1A indicates an important role in heat tolerance in potato

    Science.gov (United States)

    Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protei...

  19. Overexpression of protein kinase STK25 in mice exacerbates ectopic lipid accumulation, mitochondrial dysfunction and insulin resistance in skeletal muscle

    DEFF Research Database (Denmark)

    Chursa, Urszula; Nuñez-Durán, Esther; Cansby, Emmelie

    2017-01-01

    AIMS/HYPOTHESIS: Understanding the molecular networks controlling ectopic lipid deposition and insulin responsiveness in skeletal muscle is essential for developing new strategies to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator...... in skeletal muscle, highlighting the potential of STK25 antagonists for type 2 diabetes treatment....

  20. Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I.

    Science.gov (United States)

    Strand, Deserah D; Livingston, Aaron K; Satoh-Cruz, Mio; Koepke, Tyson; Enlow, Heather M; Fisher, Nicholas; Froehlich, John E; Cruz, Jeffrey A; Minhas, Deepika; Hixson, Kim K; Kohzuma, Kaori; Lipton, Mary; Dhingra, Amit; Kramer, David M

    2016-01-01

    We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 ( hcef2 ) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force ( pmf ), activation of the photoprotective q E response, and the accumulation of H 2 O 2 . Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H 2 O 2 in hcef2 , which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H 2 O 2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H 2 O 2 , which in turn activates CEF. This activation could be from either H 2 O 2 acting as a redox signal, or by a secondary effect from H 2 O 2 inducing a deficit in ATP.

  1. Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I

    Energy Technology Data Exchange (ETDEWEB)

    Strand, Deserah D.; Livingston, Aaron K.; Satoh-Cruz, Mio; Koepke, Tyson; Enlow, Heather M.; Fisher, Nicholas; Froehlich, John E.; Cruz, Jeffrey A.; Minhas, Deepika; Hixson, Kim K.; Kohzuma, Kaori; Lipton, Mary; Dhingra, Amit; Kramer, David M.

    2017-01-13

    We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2 . Surprisingly, hcef2 was mapped to a nonsense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDHrelated CEF. We observed similar increases in CEF and H2O2 accumulation in other translation defective mutants, suggesting that loss of coordination in plastid protein levels lead to imbalances in the photosynthetic energy balance that leads to increased CEF. These results, together with a large body of previous observations, support a general model in which processes that imbalances in chloroplast energetics result in the production of H2O2 , which activates CEF, either as a redox signal or by inducing deficits in ATP levels.

  2. CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

    Directory of Open Access Journals (Sweden)

    Hong-Wai Tham

    2014-12-01

    Full Text Available Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV. To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H screenings against DENV2 envelope (E protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1 was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  3. The prognostic significance of accumulation of p53 protein in stage III non-small cell lung cancer treated by radiotherapy

    International Nuclear Information System (INIS)

    Langendijk, J.A.; Thunnissen, F.B.J.M.; Lamers, R.J.S.; Jong, J.M.A. de; Velde, G.P.M. ten; Wouters, E.F.M.

    1995-01-01

    In the present study the prognostic significance of accumulation of nuclear p53 protein on survival and freedom from local progression was investigated. Formalin-fixed, paraffin-embedded sections obtained by bronchoscopy or mediastinoscopy were used to examine the expression of nuclear p53 protein using immunohistochemistry. In 37 cases (57%), overexpression of the p53 protein was detected. No relation was found between p53 expression and other pretreatment variables. Response to radiotherapy was found in 11 p53-negative cases (65%) versus 10 p53-positive cases (42%). Freedom from local progression was significantly better in the p53-negative cases as compared with the p53-positive cases. The p53-negative cases who responded to radiotherapy showed an excellent freedom from local progression rate after 2 years of 100%, whereas all p53-positive cases without response to radiotherapy showed local progression within 24 months. Overall survival between p53-negative and -positive cases did not differ, however the disease-specific survival was found to be worse in the p53-positive cases as compared to the negative cases (median survival 8.4 vs. 14.4 months (P < 0.05)). No correlation was found between p53 expression and the frequency of distant metastases. In conclusion, the results of this study suggest that p53 protein expression may be of prognostic value on freedom from local progression in non-small cell lung carcinoma

  4. Evaluation and Characterization of Fasciola hepatica Tegument Protein Extract for Serodiagnosis of Human Fascioliasis

    Science.gov (United States)

    Morales, Adelaida

    2012-01-01

    Tegument protein extract from Fasciola hepatica adult flukes (FhTA) was obtained and assessed for its potential as a diagnostic agent for the serological detection of human fascioliasis using an indirect enzyme-linked immunosorbent assay (ELISA). In an analysis of sera from 45 patients infected with F. hepatica, sera from 41 patients with other parasitic infections, and sera from 33 healthy controls, the FhTA-ELISA showed sensitivity, specificity, and accuracy of 91.1%, 97.3%, and 95%, respectively. Specific IgG1 and IgG4 were the antibody isotypes mainly detected in sera from patients with fascioliasis. Polypeptides of 52, 38, 24 to 26, and 12 to 14 kDa were identified by Western blotting as the most immunoreactive components of the FhTA. A proteomic approach led us to identify enolase, aldolase, glutathione S-transferase, and fatty acid binding protein as the major immunoreactive components of the FhTA. PMID:23015645

  5. Bioisosteric Replacements Extracted from High-Quality Structures in the Protein Databank.

    Science.gov (United States)

    Seddon, Matthew P; Cosgrove, David A; Gillet, Valerie J

    2018-03-20

    Bioisosterism is an important concept in the lead optimisation phase of drug discovery where the aim is to make modifications to parts of a molecule in order to improve some properties while maintaining others. We present an analysis of bioisosteric fragments extracted from the ligands in an established data set consisting of 121 protein targets. A pairwise analysis is carried out of all ligands for a given target. The ligands are fragmented using the BRICS fragmentation scheme and a pair of fragments is deemed to be bioisosteric if they occupy a similar volume of the protein binding site. We consider two levels of generality, one which does not consider the number of attachment points in the fragments and a more restricted case in which both fragments are required to have the same number of attachments. We investigate the extent to which the bioisosteric pairs that are found are common across different target. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Magnetic graphene oxide modified with choline chloride-based deep eutectic solvent for the solid-phase extraction of protein

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-02

    Highlights: • A strategy for extraction of protein based on DES-coated magnetic graphene oxide. • The deep eutectic solvents were based on choline chloride. • Bovine serum albumin was used as the analyte. • The material prepared works for the acidic but not the basic or the neutral proteins. - Abstract: Four kinds of green deep eutectic solvents (DESs) based on choline chloride (ChCl) have been synthesized and coated on the surface of magnetic graphene oxide (Fe{sub 3}O{sub 4}@GO) to form Fe{sub 3}O{sub 4}@GO-DES for the magnetic solid-phase extraction of protein. X-ray diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FTIR), field emission scanning electron microscopy (FESEM) and thermal gravimetric analysis (TGA) were employed to characterize Fe{sub 3}O{sub 4}@GO-DES, and the results indicated the successful preparation of Fe{sub 3}O{sub 4}@GO-DES. The UV–vis spectrophotometer was used to measure the concentration of protein after extraction. Single factor experiments proved that the extraction amount was influenced by the types of DESs, solution temperature, solution ionic strength, extraction time, protein concentration and the amount of Fe{sub 3}O{sub 4}@GO-DES. Comparison of Fe{sub 3}O{sub 4}@GO and Fe{sub 3}O{sub 4}@GO-DES was carried out by extracting bovine serum albumin, ovalbumin, bovine hemoglobin and lysozyme. The experimental results showed that the proposed Fe{sub 3}O{sub 4}@GO-DES performs better than Fe{sub 3}O{sub 4}@GO in the extraction of acidic protein. Desorption of protein was carried out by eluting the solid extractant with 0.005 mol L{sup −1} Na{sub 2}HPO{sub 4} contained 1 mol L{sup −1} NaCl. The obtained elution efficiency was about 90.9%. Attributed to the convenient magnetic separation, the solid extractant could be easily recycled.

  7. Inhibitory effect of Piper betle Linn. leaf extract on protein glycation--quantification and characterization of the antiglycation components.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Chakraborti, Abhay Sankar

    2013-12-01

    Piper betle Linn. is a Pan-Asiatic plant having several beneficial properties. Protein glycation and advanced glycation end products (AGEs) formation are associated with different pathophysiological conditions, including diabetes mellitus. Our study aims to find the effect of methanolic extract of P. betle leaves on in vitro protein glycation in bovine serum albumin (BSA)-glucose model. The extract inhibits glucose-induced glycation, thiol group modification and carbonyl formation in BSA in dose-dependent manner. It inhibits different stages of protein glycation, as demonstrated by using glycation models: hemoglobin-delta-gluconolactone (for early stage, Amadori product formation), BSA-methylglyoxal (for middle stage, formation of oxidative cleavage products) and BSA-glucose (for last stage, formation of AGEs) systems. Several phenolic compounds are isolated from the extract. Considering their relative amounts present in the extract, rutin appears to be the most active antiglycating agent. The extract of P. betle leaf may thus have beneficial effect in preventing protein glycation and associated complications in pathological conditions.

  8. PROTEIN L-ISOASPARTYL METHYLTRANSFERASE2 is differentially expressed in chickpea and enhances seed vigor and longevity by reducing abnormal isoaspartyl accumulation predominantly in seed nuclear proteins.

    Science.gov (United States)

    Verma, Pooja; Kaur, Harmeet; Petla, Bhanu Prakash; Rao, Venkateswara; Saxena, Saurabh C; Majee, Manoj

    2013-03-01

    PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal l-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT2') differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

  9. The potentiation of Mangifera casturi bark extract on interleukin- 1β and bone morphogenic protein-2 expressions during bone remodeling after tooth extraction

    Directory of Open Access Journals (Sweden)

    Bayu Indra Sukmana

    2017-03-01

    Full Text Available Background: The main oral health problem in Indonesia is the high number of tooth decay. Tooth extraction is the treatment often received by patients who experience tooth decay and the wound caused by alveolar bone resorption. Bark of Mangifera casturi has been studied and proven to contain secondary metabolite which has the ability to increase osteoblast’s activity and suppress osteoclast’s activity. Purpose: The purpose of this study was to analyze interleukin-1 beta (IL-1β and bone morphogenic protein-2 (BMP-2 activities during bone remodeling after Mangifera casturi’s bark extract treatment. Method: This study was laboratory experimental research with randomized post-test only control group design. The Mangifera casturi bark was extracted using 96% ethanol maceration and n-hexane fractionation. This study used 40 male Wistar rats which are divided into 4 groups and the tooth extraction was performed on the rats’ right mandible incisive tooth. The four groups consisted of 6.35%, 12.7%, 25.4% extract treatment group, and a control group. Wistar’s mandibles were decapitated on the 7th and 14th day after extraction. Antibody staining on preparations for the examination of IL-1β and BMP-2 expressions was done using immunohistochemistry. Result: There was a significant difference of IL-1β and BMP-2 expressions in 6,35%, 12,7%, and 25,4% treatment groups compared to control group with p<0.05. Conclusion: Mangifera casturi’s bark extract was able to suppress the IL-1β expression and increase the BMP-2 expression during bone remodeling after tooth extraction.

  10. 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...... for the use as animal feed. The residual 90% of organic matter, leaving the separation as solid press cake and brown juice was subjected to anaerobic digestion to produce biogas and fertilizer. Methane yields of 220-310 and 430-540 ml CH4/g VS were obtained for press cake and brown juice, respectively...

  11. Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

    Science.gov (United States)

    Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

    2006-02-01

    The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

  12. LOX Gene Transcript Accumulation in Olive (Olea europaea L. Fruits at Different Stages of Maturation: Relationship between Volatile Compounds, Environmental Factors, and Technological Treatments for Oil Extraction

    Directory of Open Access Journals (Sweden)

    Innocenzo Muzzalupo

    2012-01-01

    Full Text Available The quality of olive oil is influenced by genetic and environmental factors and by the maturation state of drupes, but it is equally affected by technological treatments of the process. This work investigates the possible correlation between olive LOX gene transcript accumulation, evaluated in fruits collected at different stages of maturation, and chemical biomarkers of its activity. During olive fruit ripening, the same genotype harvested from two different farms shows a positive linear trend between LOX relative transcript accumulation and the content of volatile compounds present in the olive oil aroma. Interestingly, a negative linear trend was observed between LOX relative transcript accumulation and the content of volatile compounds present in the olive pastes obtained from olive fruits with and without malaxation. The changes in the olive LOX transcript accumulation reveal its environmental regulation and suggest differential physiological functions for the LOXs.

  13. Alkylresorcinols in rye (Secale cereale L. caryopses. III. Application of detergents for extraction of proteins and alkylresorcinols

    Directory of Open Access Journals (Sweden)

    W. Mejbaum-Katzenellenbogen

    2015-01-01

    Full Text Available In the work here described sodium dodecyl sulphate solutions (SDS, tween 80 and triton X 100 were used for isolation of proteins and 5-n-alkylresorcinols from ground rye grain. It was found that the above named detergents extract different protein and various amounts of alkylresorcin derivatives. The results indicate that 5-n-alkylresorcinols are localized in the membraneous structures of rye caryopses.

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

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

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

  15. Cerebral amyloid-beta protein accumulation with aging in cotton-top tamarins: a model of early Alzheimer's disease?

    Science.gov (United States)

    Lemere, Cynthia A; Oh, Jiwon; Stanish, Heather A; Peng, Ying; Pepivani, Imelda; Fagan, Anne M; Yamaguchi, Haruyasu; Westmoreland, Susan V; Mansfield, Keith G

    2008-04-01

    Alzheimer's disease (AD) is the most common progressive form of dementia in the elderly. Two major neuropathological hallmarks of AD include cerebral deposition of amyloid-beta protein (Abeta) into plaques and blood vessels, and the presence of neurofibrillary tangles in brain. In addition, activated microglia and reactive astrocytes are often associated with plaques and tangles. Numerous other proteins are associated with plaques in human AD brain, including Apo E and ubiquitin. The amyloid precursor protein and its shorter fragment, Abeta, are homologous between humans and non-human primates. Cerebral Abeta deposition has been reported previously for rhesus monkeys, vervets, squirrel monkeys, marmosets, lemurs, cynomologous monkeys, chimpanzees, and orangutans. Here we report, for the first time, age-related neuropathological changes in cotton-top tamarins (CTT, Saguinus oedipus), an endangered non-human primate native to the rainforests of Colombia and Costa Rica. Typical lifespan is 13-14 years of age in the wild and 15-20+ years in captivity. We performed detailed immunohistochemical analyses of Abeta deposition and associated pathogenesis in archived brain sections from 36 tamarins ranging in age from 6-21 years. Abeta plaque deposition was observed in 16 of the 20 oldest tamarins (>12 years). Plaques contained mainly Abeta42, and in the oldest animals, were associated with reactive astrocytes, activated microglia, Apo E, and ubiquitin-positive dystrophic neurites, similar to human plaques. Vascular Abeta was detected in 14 of the 20 aged tamarins; Abeta42 preceded Abeta40 deposition. Phospho-tau labeled dystrophic neurites and tangles, typically present in human AD, were absent in the tamarins. In conclusion, tamarins may represent a model of early AD pathology.

  16. Magnolol Alleviates Inflammatory Responses and Lipid Accumulation by AMP-Activated Protein Kinase-Dependent Peroxisome Proliferator-Activated Receptor α Activation

    Directory of Open Access Journals (Sweden)

    Ye Tian

    2018-02-01

    Full Text Available Magnolol (MG is a kind of lignin isolated from Magnolia officinalis, which serves several different biological functions, such as antifungal, anticancer, antioxidant, and hepatoprotective functions. This study aimed to evaluate the protective effect of MG against oleic acid (OA-induced hepatic steatosis and inflammatory damage in HepG2 cells and in a tyloxapol (Ty-induced hyperlipidemia mouse model. Our findings indicated that MG can effectively inhibit OA-stimulated tumor necrosis factor α (TNF-α secretion, reactive oxygen species generation, and triglyceride (TG accumulation. Further study manifested that MG significantly suppressed OA-activated mitogen-activated protein kinase (MAPK and nuclear factor-kappa B (NF-κB signaling pathways and that these inflammatory responses can be negated by pretreatment with inhibitors of extracellular regulated protein kinase and c-Jun N-terminal kinase (U0126 and SP600125, respectively. In addition, MG dramatically upregulated peroxisome proliferator-activated receptor α (PPARα translocation and reduced sterol regulatory element-binding protein 1c (SREBP-1c protein synthesis and excretion, both of which are dependent upon the phosphorylation of adenosine monophosphate (AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase, and AKT kinase (AKT. However, MG suspended the activation of PPARα expression and was thus blocked by pretreatment with LY294002 and compound c (specific inhibitors of AKT and AMPK. Furthermore, MG clearly alleviated serum TG and total cholesterol release; upregulated AKT, AMPK, and PPARα expression; suppressed SREBP-1c generation; and alleviated hepatic steatosis and dyslipidemia in Ty-induced hyperlipidemia mice. Taken together, these results suggest that MG exerts protective effects against steatosis, hyperlipidemia, and the underlying mechanism, which may be closely associated with AKT/AMPK/PPARα activation and MAPK/NF-κB/SREBP-1c inhibition.

  17. A study on the relationship of arsenic accumulation with protein, lipid, ash and moisture contents in muscle of eight species of fish in Iran

    Directory of Open Access Journals (Sweden)

    A Askary Sary

    2012-11-01

    Full Text Available A comparative study was conducted to investigate a relationship between concentration of arsenic with protein, lipid, ash and moisture content in Cyprinus carpio, Oncorhynchus mykiss, Aristichthys nobilis, Hypophthalmichthys molitrix, Ctenopharyngodon idella, Scomberomorus commerson, Scomberomorus guttatus and Otolithes ruber. A total of 72 sample of common carp, Bighead carp, silver carp and grass carp fishing from Azadegan fish farming center, Ahvaz; Rainbow trout from Cheshme Dimeh and Scomberomorus commerson, Scomberomorus guttatus and Otolithes ruber caught with gill netfrom Hendijan. Wet-digestion method was performed prior to arsenic determination in the samples. The level of arsenic was measured by atomic absorption spectrophotometer. The results showed that concentration of arsenic in the muscle of fishes was 269.87 ± 20.96 µg/Kg. Moreover, levels of protein, lipid, ash and moisture in the samples were estimated at 19.67±0.78 g/100, 2.45±0.45 g/100, 1.49±0.23 g/100, 78±1.89 g/100, respectively. Results also showed a positive correlation between the accumulation of arsenic in muscle of fishes with levels of protein, lipid, ash and moisture (p

  18. Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James; Takenaka, Yasuhiro; Stampfer, Martha R.; Gilley, David; Yaswen, Paul

    2004-12-23

    We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.

  19. PPI-IRO: A two-stage method for protein-protein interaction extraction based on interaction relation ontology

    KAUST Repository

    Li, Chuanxi; Chen, Peng; Wang, Rujing; Wang, Xiujie; Su, Yaru; Li, Jinyan

    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

  20. Effect of Plant Age on the Quantity and Quality of Proteins Extracted from Sugar Beet (Beta vulgaris L.) Leaves

    NARCIS (Netherlands)

    Kiskini, Alexandra; Vissers, Anne; Vincken, Jean Paul; Gruppen, Harry; Wierenga, Peter Alexander

    2016-01-01

    Effects of the developmental stage (e.g., young, mature, or senescent) of leaves on their chemical composition have been described in the literature. This study focuses on the variation in chemical composition and quantity and quality of proteins extracted from leaves due to variation in plant

  1. Investigating the microstructure of keratin extracted from wool: peptide sequence (MALDI-TOF/TOF) and protein conformation (FTIR)

    Science.gov (United States)

    Keratin was extracted from wool by reduction with 2-mercaptoethanol. It was isolated as intact keratin and characterized by its similar molecular weight, protein composition, and secondary structure to native keratin. Gel electrophoresis patterns and MALDI-TOF/TOF peptide sequences provided the ide...

  2. Antioxidative Effect of Seaweed Extracts in Chilled Storage of Minced Atlantic Mackerel (Scomber scombrus): Effect on Lipid and Protein Oxidation

    DEFF Research Database (Denmark)

    Babakhani, Aria; Farvin, K. H Sabeena; Jacobsen, Charlotte

    2016-01-01

    In this study, antioxidant activity of absolute ethanol, 50 % ethanol and water extracts of two species of seaweeds namely, Fucus serratus and Polysiphonia fucoides were evaluated for their ability to retard lipid and protein oxidation in minced mackerel. Mackerel mince added with 0.5 g/kg of ext...

  3. Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

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    Carol B Fowler

    2010-12-01

    Full Text Available Proteomic studies of formalin-fixed paraffin-embedded (FFPE tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%. Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

  4. Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

    Science.gov (United States)

    Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

    2010-01-01

    Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form

  5. Data on amyloid precursor protein accumulation, spontaneous physical activity, and motor learning after traumatic brain injury in the triple-transgenic mouse model of Alzheimer׳s disease

    Directory of Open Access Journals (Sweden)

    Yasushi Kishimoto

    2016-12-01

    Full Text Available This data article contains supporting information regarding the research article entitled “Traumatic brain injury accelerates amyloid-β deposition and impairs spatial learning in the triple-transgenic mouse model of Alzheimer׳s disease” (H. Shishido, Y. Kishimoto, N. Kawai, Y. Toyota, M. Ueno, T. Kubota, Y. Kirino, T. Tamiya, 2016 [1]. Triple-transgenic (3×Tg-Alzheimer׳s disease (AD model mice exhibited significantly poorer spatial learning than sham-treated 3×Tg-AD mice 28 days after traumatic brain injury (TBI. Correspondingly, amyloid-β (Aβ deposition within the hippocampus was significantly greater in 3×Tg-AD mice 28 days after TBI. However, data regarding the short-term and long-term influences of TBI on amyloid precursor protein (APP accumulation in AD model mice remain limited. Furthermore, there is little data showing whether physical activity and motor learning are affected by TBI in AD model mice. Here, we provide immunocytochemistry data confirming that TBI induces significant increases in APP accumulation in 3×Tg-AD mice at both 7 days and 28 days after TBI. Furthermore, 3×Tg-AD model mice exhibit a reduced ability to acquire conditioned responses (CRs during delay eyeblink conditioning compared to sham-treated 3×Tg-AD model mice 28 days after TBI. However, physical activity and motor performance are not significantly changed in TBI-treated 3×Tg-AD model mice.

  6. Pu-Erh Tea Extract Induces the Degradation of FET Family Proteins Involved in the Pathogenesis of Amyotrophic Lateral Sclerosis

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    Yang Yu

    2014-01-01

    Full Text Available FET family proteins consist of fused in sarcoma/translocated in liposarcoma (FUS/TLS, Ewing's sarcoma (EWS, and TATA-binding protein-associated factor 15 (TAF15. Mutations in the copper/zinc superoxide dismutase (SOD1, TAR DNA-binding protein 43 (TDP-43, and FET family proteins are associated with the development of amyotrophic lateral sclerosis (ALS, a fatal neurodegenerative disease. There is currently no cure for this disease and few effective treatments are available. Epidemiological studies indicate that the consumption of tea is associated with a reduced risk of developing neurodegenerative diseases. The results of this study revealed that components of a pu-erh tea extract (PTE interacted with FET family proteins but not with TDP-43 or SOD1. PTE induced the degradation of FET family proteins but had no effects on TDP-43 or SOD1. The most frequently occurring ALS-linked FUS/TLS mutant protein, R521C FUS/TLS, was also degraded in the presence of PTE. Furthermore, ammonium chloride, a lysosome inhibitor, but not lactacystin, a proteasome inhibitor, reduced the degradation of FUS/TLS protein by PTE. PTE significantly reduced the incorporation of R521C FUS/TLS into stress granules under stress conditions. These findings suggest that PTE may have beneficial health effects, including preventing the onset of FET family protein-associated neurodegenerative diseases and delaying the progression of ALS by inhibiting the cytoplasmic aggregation of FET family proteins.

  7. Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

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    Adi Ben Yehuda

    Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

  8. The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate Jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana.

    Science.gov (United States)

    Qi, Tiancong; Song, Susheng; Ren, Qingcuo; Wu, Dewei; Huang, Huang; Chen, Yan; Fan, Meng; Peng, Wen; Ren, Chunmei; Xie, Daoxin

    2011-05-01

    Jasmonates (JAs) mediate plant responses to insect attack, wounding, pathogen infection, stress, and UV damage and regulate plant fertility, anthocyanin accumulation, trichome formation, and many other plant developmental processes. Arabidopsis thaliana Jasmonate ZIM-domain (JAZ) proteins, substrates of the CORONATINE INSENSITIVE1 (COI1)-based SCF(COI1) complex, negatively regulate these plant responses. Little is known about the molecular mechanism for JA regulation of anthocyanin accumulation and trichome initiation. In this study, we revealed that JAZ proteins interact with bHLH (Transparent Testa8, Glabra3 [GL3], and Enhancer of Glabra3 [EGL3]) and R2R3 MYB transcription factors (MYB75 and Glabra1), essential components of WD-repeat/bHLH/MYB transcriptional complexes, to repress JA-regulated anthocyanin accumulation and trichome initiation. Genetic and physiological evidence showed that JA regulates WD-repeat/bHLH/MYB complex-mediated anthocyanin accumulation and trichome initiation in a COI1-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors (GL3 and EGL3) restored anthocyanin accumulation and trichome initiation in the coi1 mutant, respectively. We speculate that the JA-induced degradation of JAZ proteins abolishes the interactions of JAZ proteins with bHLH and MYB factors, allowing the transcriptional function of WD-repeat/bHLH/MYB complexes, which subsequently activate respective downstream signal cascades to modulate anthocyanin accumulation and trichome initiation.

  9. The Jasmonate-ZIM-Domain Proteins Interact with the WD-Repeat/bHLH/MYB Complexes to Regulate Jasmonate-Mediated Anthocyanin Accumulation and Trichome Initiation in Arabidopsis thaliana[C][W

    Science.gov (United States)

    Qi, Tiancong; Song, Susheng; Ren, Qingcuo; Wu, Dewei; Huang, Huang; Chen, Yan; Fan, Meng; Peng, Wen; Ren, Chunmei; Xie, Daoxin

    2011-01-01

    Jasmonates (JAs) mediate plant responses to insect attack, wounding, pathogen infection, stress, and UV damage and regulate plant fertility, anthocyanin accumulation, trichome formation, and many other plant developmental processes. Arabidopsis thaliana Jasmonate ZIM-domain (JAZ) proteins, substrates of the CORONATINE INSENSITIVE1 (COI1)–based SCFCOI1 complex, negatively regulate these plant responses. Little is known about the molecular mechanism for JA regulation of anthocyanin accumulation and trichome initiation. In this study, we revealed that JAZ proteins interact with bHLH (Transparent Testa8, Glabra3 [GL3], and Enhancer of Glabra3 [EGL3]) and R2R3 MYB transcription factors (MYB75 and Glabra1), essential components of WD-repeat/bHLH/MYB transcriptional complexes, to repress JA-regulated anthocyanin accumulation and trichome initiation. Genetic and physiological evidence showed that JA regulates WD-repeat/bHLH/MYB complex-mediated anthocyanin accumulation and trichome initiation in a COI1-dependent manner. Overexpression of the MYB transcription factor MYB75 and bHLH factors (GL3 and EGL3) restored anthocyanin accumulation and trichome initiation in the coi1 mutant, respectively. We speculate that the JA-induced degradation of JAZ proteins abolishes the interactions of JAZ proteins with bHLH and MYB factors, allowing the transcriptional function of WD-repeat/bHLH/MYB complexes, which subsequently activate respective downstream signal cascades to modulate anthocyanin accumulation and trichome initiation. PMID:21551388

  10. Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

    Science.gov (United States)

    Hassig, Christian A; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E; Brown, Susan G; Baire, Beeraiah; Michel, Andrew R; Hoye, Thomas R; Francis, Subhashree; Georg, Gunda I; Walters, Michael A; Divlianska, Daniela B; Roth, Gregory P; Wright, Amy E; Reed, John C

    2014-09-01

    Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach. © 2014 Society for Laboratory Automation and Screening.

  11. Simultaneous effect of nitrate (NO3- concentration, carbon dioxide (CO2 supply and nitrogen limitation on biomass, lipids, carbohydrates and proteins accumulation in Nannochloropsis oculata

    Directory of Open Access Journals (Sweden)

    Aarón Millán-Oropeza

    2015-03-01

    Full Text Available Biodiesel from microalgae is a promising technology. Nutrient limitation and the addition of CO2 are two strategies to increase lipid content in microalgae. There are two different types of nitrogen limitation, progressive and abrupt limitation. In this work, the simultaneous effect of initial nitrate concentration, addition of CO2, and nitrogen limitation on biomass, lipid, protein and carbohydrates accumulation were analyzed. An experimental design was established in which initial nitrogen concentration, culture time and CO2 aeration as independent numerical variables with three levels were considered. Nitrogen limitation was taken into account as a categorical independent variable. For the experimental design, all the experiments were performed with progressive nitrogen limitation. The dependent response variables were biomass, lipid production, carbohydrates and proteins. Subsequently, comparison of both types of limitation i.e. progressive and abrupt limitation, was performed. Nitrogen limitation in a progressive mode exerted a greater effect on lipid accumulation. Culture time, nitrogen limitation and the interaction of initial nitrate concentration with nitrogen limitation had higher influences on lipids and biomass production. The highest lipid production and productivity were at 582 mgL-1 (49.7 % lipid, dry weight basis and 41.5 mgL-1d-1, respectively; under the following conditions: 250 mgL-1 of initial nitrate concentration, CO2 supply of 4% (v/v, 12 d of culturing and 2 d in state of nitrogen starvation induced by progressive limitation. This work presents a novel way to perform simultaneous analysis of the effect of the initial concentration of nitrate, nitrogen limitation, and CO2 supply on growth and lipid production of Nannochloropsis oculata, with the aim to produce potential biofuels feedstock.

  12. Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation.

    Science.gov (United States)

    Das, Natasha; Bhattacharya, Surajit; Maiti, Mrinal K

    2016-08-01

    One of the most grievous heavy metal pollutants in the environment is cadmium (Cd), which is not only responsible for the crop yield loss owing to its phytotoxicity, but also for the human health hazards as the toxic elements usually accumulate in the consumable parts of crop plants. In the present study, we aimed to isolate and functionally characterize the OsMTP1 gene from indica rice (Oryza sativa L. cv. IR64) to study its potential application for efficient phytoremediation of Cd. The 1257 bp coding DNA sequence (CDS) of OsMTP1 encodes a ∼46 kDa protein belonging to the cation diffusion facilitator (CDF) or metal tolerance/transport protein (MTP) family. The OsMTP1 transcript in rice plant was found to respond during external Cd stress. Heterologous expression of OsMTP1 in tobacco resulted in the reduction of Cd stress-induced phytotoxic effects, including growth inhibition, lipid peroxidation, and cell death. Compared to untransformed control, the transgenic tobacco plants showed enhanced vacuolar thiol content, indicating vacuolar localization of the sequestered Cd. The transgenic tobacco plants exhibited significantly higher biomass growth (2.2-2.8-folds) and hyperaccumulation of Cd (1.96-2.22-folds) compared to untransformed control under Cd exposure. The transgenic plants also showed moderate tolerance and accumulation of arsenic (As) upon exogenous As stress, signifying broad substrate specificity of OsMTP1. Together, findings of our research suggest that the transgenic tobacco plants overexpressing OsMTP1 with its hyperaccumulating activity and increased growth rate could be useful for future phytoremediation applications to clean up the Cd-contaminated soil. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Looking deep inside: detection of low-abundance proteins in leaf extracts of Arabidopsis and phloem exudates of pumpkin.

    Science.gov (United States)

    Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg; Lindermayr, Christian

    2012-07-01

    The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome.

  14. A Natural Antibacterial-Antioxidant Film from Soy Protein Isolate Incorporated with Cortex Phellodendron Extract

    Directory of Open Access Journals (Sweden)

    Shumin Liang

    2018-01-01

    Full Text Available An active film was prepared by incorporating cortex Phellodendron extract (CPE, an active agent into a soybean protein isolate (SPI. Different concentrations of CPE (0%, 10%, 12.5%, 15%, 17.5%, 20%, or 22.5%, w/w, based on SPI were mixed into the films characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, thermogravimetry, tensile tests, and barrier properties. The rheological properties of the solutions were also tested. The effects of the CPE content on the antibacterial and antioxidant activities of the films were examined. The results indicated that new hydrogen bonds formed between molecules in the films, and the crystallinity of the films decreased. The incorporation of CPE had no significant influence on the thermal stability of the films. Films containing 15% CPE had the maximum tensile strength of 6.00 MPa. The barrier properties against water vapor, oxygen, and light enhanced with the incorporation of CPE. The antioxidant activity of the SPI film was also improved. The films were effective against Staphylococcus aureus (S. aureus, Gram-positive bacteria. These results suggest that the SPI/CPE film can potentially extend the shelf lives of foods.

  15. Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Olena Zakharchenko

    2011-01-01

    Full Text Available Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE. This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

  16. Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats.

    Science.gov (United States)

    Iamsaard, Sitthichai; Prabsattroo, Thawatchai; Sukhorum, Wannisa; Muchimapura, Supaporn; Srisaard, Panee; Uabundit, Nongnut; Thukhammee, Wipawee; Wattanathorn, Jintanaporn

    2013-03-01

    To investigate the effect of Anethum graveolens (AG) extracts on the mounting frequency, histology of testis and epididymis, and sperm physiology. Male rats induced by cold immobilization before treating with vehicle or AG extracts [50, 150, and 450 mg/kg body weight (BW)] via gastric tube for consecutive 1, 7, and 14 d were examined for mounting frequency, testicular phosphorylation level by immunoblotting, sperm concentration, sperm acrosome reaction, and histological structures of testis and epididymis, respectively. AG (50 mg/kg BW) significantly increased the mounting frequency on Days 1 and 7 compared to the control group. Additionally, rat testis treated with 50 mg/kg BW AG showed high levels of phosphorylated proteins as compared with the control group. In histological analyses, AG extract did not affect the sperm concentration, acrosome reaction, and histological structures of testis and epididymis. AG extract enhances the aphrodisiac activity and is not harmful to sperm and male reproductive organs.

  17. Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats*

    Science.gov (United States)

    Iamsaard, Sitthichai; Prabsattroo, Thawatchai; Sukhorum, Wannisa; Muchimapura, Supaporn; Srisaard, Panee; Uabundit, Nongnut; Thukhammee, Wipawee; Wattanathorn, Jintanaporn

    2013-01-01

    Objective: To investigate the effect of Anethum graveolens (AG) extracts on the mounting frequency, histology of testis and epididymis, and sperm physiology. Methods: Male rats induced by cold immobilization before treating with vehicle or AG extracts [50, 150, and 450 mg/kg body weight (BW)] via gastric tube for consecutive 1, 7, and 14 d were examined for mounting frequency, testicular phosphorylation level by immunoblotting, sperm concentration, sperm acrosome reaction, and histological structures of testis and epididymis, respectively. Results: AG (50 mg/kg BW) significantly increased the mounting frequency on Days 1 and 7 compared to the control group. Additionally, rat testis treated with 50 mg/kg BW AG showed high levels of phosphorylated proteins as compared with the control group. In histological analyses, AG extract did not affect the sperm concentration, acrosome reaction, and histological structures of testis and epididymis. Conclusions: AG extract enhances the aphrodisiac activity and is not harmful to sperm and male reproductive organs. PMID:23463768

  18. Extraction of Pathogenesis-Related Proteins and Phenolics in Sauvignon Blanc as Affected by Grape Harvesting and Processing Conditions

    Directory of Open Access Journals (Sweden)

    Bin Tian

    2017-07-01

    Full Text Available Thaumatin-like proteins (TLPs and chitinases are the two main groups of pathogenesis-related (PR proteins found in wine that cause protein haze formation. Previous studies have found that phenolics are also involved in protein haze formation. In this study, Sauvignon Blanc grapes were harvested and processed in two vintages (2011 and 2012 by three different treatments: (1 hand harvesting with whole bunch press (H-WB; (2 hand harvesting with destem/crush and 3 h skin contact (H-DC-3; and (3 machine harvesting with destem/crush and 3 h skin contact (M-DC-3. The juices were collected at three pressure levels (0.4 MPa, 0.8 MPa and 1.6 MPa, some juices were fermented in 750 mL of wine bottles to determine the bentonite requirement for the resulting wines. Results showed juices of M-DC-3 had significantly lower concentration of proteins, including PR proteins, compared to those of H-DC-3, likely due to the greater juice yield of M-DC-3 and interactions between proteins and phenolics. Juices from the 0.8–1.6 MPa pressure and resultant wines had the highest concentration of phenolics but the lowest concentration of TLPs. This supported the view that TLPs are released at low pressure as they are mainly present in grape pulp but additional extraction of phenolics largely present in skin occurs at higher pressing pressure. Wine protein stability tests showed a positive linear correlation between bentonite requirement and the concentration of chitinases, indicating the possibility of predicting bentonite requirement by quantification of chitinases. This study contributes to an improved understanding of extraction of haze-forming PR proteins and phenolics that can influence bentonite requirement for protein stabilization.

  19. Inhibitory effect of Clitoria ternatea flower petal extract on fructose-induced protein glycation and oxidation-dependent damages to albumin in vitro.

    Science.gov (United States)

    Chayaratanasin, Poramin; Barbieri, Manuel Alejandro; Suanpairintr, Nipattra; Adisakwattana, Sirichai

    2015-02-18

    The accumulation of advanced glycation end products (AGEs) in body tissue has been implicated in the progression of age-related diseases. Inhibition of AGE formation is the imperative approach for alleviating diabetic complications. Clitoria ternatea extract (CTE) has been demonstrated to possess anti-diabetic activity. However, there is no scientific evidence supporting its anti-glycation activity. The objective of this study was to determine the inhibitory effect of CTE on fructose-induced formation of AGEs and protein oxidation. Antioxidant activity of CTE was also assessed by various methods. The aqueous extract of CTE (0.25-1.00 mg/ml) was measured for the content of total phenolic compounds, flavonoid, and anthocyanin by Folin-Ciocalteu assay, AlCl3 colorimetric method, and pH differential method, respectively. The various concentrations of CTE were incubated with BSA and fructose at 37°C for 28 days. The formation of fluorescent AGEs, the level of fructosamine, protein carbonyl content, and thiol group were measured. The in vitro antioxidant activity was measured by the 1,1-diphenyl 2-picrylhydrazyl (DPPH) scavenging activity, trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), hydroxyl radical scavenging activity (HRSA), superoxide radical scavenging activity (SRSA), and ferrous ion chelating power (FICP). The results demonstrated that the content of total phenolics, flavonoids and total anthocyanins in CTE was 53 ± 0.34 mg gallic acid equivalents/g dried extract, 11.2 ± 0.33 mg catechin equivalents/g dried extract, and 1.46 ± 0.04 mg cyanidin-3-glucoside equivalents/g dried extract, respectively. Moreover, CTE (0.25-1.00 mg/ml) significantly inhibited the formation of AGEs in a concentration-dependent manner. CTE also markedly reduced the levels of fructosamine and the oxidation of protein by decreasing protein carbonyl content and preventing free thiol depletion. In the DPPH radical scavenging

  20. Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Precursor Protein Transgenic Mice

    Science.gov (United States)

    Liu, Peng; Reichl, John H.; Rao, Eshaan R.; McNellis, Brittany M.; Huang, Eric S.; Hemmy, Laura S.; Forster, Colleen L.; Kuskowski, Michael A.; Borchelt, David R.; Vassar, Robert; Ashe, Karen H.; Zahs, Kathleen R.

    2016-01-01

    There exist several dozen lines of transgenic mice that express human amyloid-β precursor protein (AβPP) with Alzheimer’s disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ~4.5 times that of 21-month Tg2576 mice and ~15 times that of 21–24-month rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort. PMID:28059792

  1. Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Protein Precursor Transgenic Mice.

    Science.gov (United States)

    Liu, Peng; Reichl, John H; Rao, Eshaan R; McNellis, Brittany M; Huang, Eric S; Hemmy, Laura S; Forster, Colleen L; Kuskowski, Michael A; Borchelt, David R; Vassar, Robert; Ashe, Karen H; Zahs, Kathleen R

    2017-01-01

    There exist several dozen lines of transgenic mice that express human amyloid-β protein precursor (AβPP) with Alzheimer's disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ∼4.5 times that of 21-month-old Tg2576 mice and ∼15 times that of 21-24-month-old rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort.

  2. Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

    Science.gov (United States)

    Nguyen-Khuong, Terry; White, Melanie Y; Hung, Tzong-Tyng; Seeto, Shona; Thomas, Melissa L; Fitzgerald, Anna M; Martucci, Carlos E; Luk, Sharon; Pang, Shiu-Fu; Russell, Pamela J; Walsh, Bradley J

    2009-04-01

    Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

  3. SA-Mot: a web server for the identification of motifs of interest extracted from protein loops.

    Science.gov (United States)

    Regad, Leslie; Saladin, Adrien; Maupetit, Julien; Geneix, Colette; Camproux, Anne-Claude

    2011-07-01

    The detection of functional motifs is an important step for the determination of protein functions. We present here a new web server SA-Mot (Structural Alphabet Motif) for the extraction and location of structural motifs of interest from protein loops. Contrary to other methods, SA-Mot does not focus only on functional motifs, but it extracts recurrent and conserved structural motifs involved in structural redundancy of loops. SA-Mot uses the structural word notion to extract all structural motifs from uni-dimensional sequences corresponding to loop structures. Then, SA-Mot provides a description of these structural motifs using statistics computed in the loop data set and in SCOP superfamily, sequence and structural parameters. SA-Mot results correspond to an interactive table listing all structural motifs extracted from a target structure and their associated descriptors. Using this information, the users can easily locate loop regions that are important for the protein folding and function. The SA-Mot web server is available at http://sa-mot.mti.univ-paris-diderot.fr.

  4. Smoke Priming, a Potent Protective Agent Against Salinity: Effect on Proline Accumulation, Elemental Uptake, Pigmental Attributes and Protein Banding Patterns of Rice (Oryza Sativa

    Directory of Open Access Journals (Sweden)

    Jamil, Muhammad

    2013-02-01

    Full Text Available The exogenous application of plant derived smoke solution through seed pre treatment is consider to create tolerance in the plant against salinity, for this purpose different dilution of plant derived smoke solution as 1:5000 Buhania, 1:1000 Buhania, 1:1000 Cymbopogon, 1:500 Cymbopogon were used against 0 mM, 50, 100 and 150mM NaCl solution in the medium. The effect was observed on total proline accumulation, heavy metals uptake, photosynthetic pigments and protein polypeptide bands intensity in two rice varieties as Basmati 385 (B-385 and Shaheen Basmati (S. Basmati. Proline concentration increases while chlorophyll “a” chlorophyll “b” and carotene level decreases with increasing salinity. On other hand zinc concentration increases while cadmium and lead concentration decrease in the crop under saline conditions. Intensity of protein polypeptides bands decreases gradually with increasing salinity level but plants from the seeds soaked with smoke solution alleviate the drastic affect of salinity, and intensity of bands is quite good by comparing with non primed seeds. It is concluded that seed priming with plant derived smoke solution show beneficial effect on crop to protect them from salinity.

  5. Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

    Science.gov (United States)

    Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

    2017-01-01

    In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

  6. Nepeta Dschuparensis Bornm Extract Moderates COX-2 and IL-1β Proteins in a Rat Model of Cerebral Ischemia

    Directory of Open Access Journals (Sweden)

    Alireza Mousavi Nia

    2017-03-01

    Full Text Available Background: Nepeta dschuparensis Bornm (NP is used as a medicinal herb in Iran. In traditional medicine, this herb is extensively employed for curing ailments such as cardiovascular diseases. NP has antioxidant and anti-inflammatory properties. This project examined the effects of the NP extract on cyclooxygenase-2 (COX-2 and interleukin-1β (IL-1β protein levels and its efficacy in neuroprotection in a cerebral ischemia-reperfusion model. Methods: Twenty-six male rats were randomly divided into 3 groups: 1 sham (n=6: no middle cerebral artery occlusion (MCAO procedure, 2 control (n=10: MCAO procedure and treatment with normal saline, and 3 NP extract (n=10: MCAO procedure and treatment with the NP extract (20 mg/kg, i.p. at the beginning of reperfusion. To examine the injury caused by cerebral ischemia, we measured motor coordination and the infarct area using the rotarod test and triphenyl tetrazolium chloride staining, respectively. IL-1β and COX-2 protein levels, as inflammatory markers, were measured by immunoblotting assay. The statistical analyses were performed using SPSS, version 16, and the data are expressed as means±SEMs. Statistical difference was evaluated using the one-way ANOVA, followed by the post hoc LSD test (P<0.01. Results: Treatment with the NP extract significantly diminished the infarct volume and alleviated the motor coordination disorder induced by cerebral ischemia. The NP extract administration significantly attenuated the increase in IL-1β and COX-2 protein levels too (P<0.01. Conclusion: The beneficial effects of the NP extract are related to its ability to decrease the levels of IL-1β and COX-2.

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

    OpenAIRE

    V Vengaiah; A Govardhan Naik; C Changamma

    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, esti...

  8. Allergen extracts and recombinant proteins: comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.

    Science.gov (United States)

    Smoldovskaya, Olga; Feyzkhanova, Guzel; Arefieva, Alla; Voloshin, Sergei; Ivashkina, Olga; Reznikov, Yuriy; Rubina, Alla

    2016-01-01

    Immunological test systems for diagnostics of type I hypersensitivity involve the following types of antigens: whole allergen extracts, individual highly purified proteins and their recombinant analogues. The goal of this study was to compare the results obtained with whole allergen extracts (birch pollen, cat dander, and timothy grass pollen) and their respective recombinant proteins in biochip-based immunoassay. Multiplex fluorescent immunoassay of 139 patients' blood serum samples was carried out using biological microchips (biochips). sIgE concentrations for the chosen allergens and their recombinant components were measured. ROC analysis was used for comparison of the results and determination of diagnostic accuracy. The results for the birch pollen extract and its recombinant allergens have shown that the diagnostic accuracy of the methods utilizing the whole allergen extract, its major component Bet v 1 and the combination of major and minor components (Bet v 1 and Bet v 2) was the same. Values for diagnostic accuracy for the cat dander extract and its major recombinant component Fel d 1 were equal. In contrast with birch pollen and cat dander allergens, using of recombinant components of timothy grass pollen (Phl p 1, Phl p 5, Phl p 7 and Phl p 12) did not allow reaching the diagnostic accuracy of using natural extract. Multiplex analysis of samples obtained from patients with allergy to birch pollen and cat dander using biological microchips has shown that comparable accuracy was observed for the assay with natural extracts and recombinant allergens. In the case of timothy grass allergen, using the recombinant components may be insufficient.

  9. Antiatherosclerotic Effect of Korean Red Ginseng Extract Involves Regulator of G-Protein Signaling 5

    Directory of Open Access Journals (Sweden)

    Eun Ju Im

    2014-01-01

    Full Text Available Regulator of G-protein signaling 5 (RGS5, an inhibitor of Gα(q and Gα(i activation, has been reported to have antiatherosclerosis. Previous studies showed antiatherosclerotic effect of Korean red ginseng water extract (KRGE via multiple signaling pathways. However, potential protective effect of KRGE through RGS5 expression has not been elucidated. Here, we investigated the antiatherosclerotic effect of KRGE in vivo and in vitro and its role on RGS5 mRNA expression. Elevated levels of total cholesterol, lactate dehydrogenase (LDH, and triglyceride (TG in western diet groups of low-density lipoprotein receptor deficient LDLr−/− mice were reversed by oral administration of KRGE. KRGE suppressed transcriptional activity of tumor necrotic factor alpha (TNF-α, interleukin-6 (IL-6, and leptin in adipose tissue. It also potently repressed western diet-induced atheroma formation in aortic sinus. While KRGE showed reduced mRNA expression of inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (COX-2, IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells, it enhanced mRNA expression of RGS5. Moreover, RGS5 siRNA transfection of microglia cells pretreated with KRGE reversed its inhibitory effect on the expression of iNOS, COX-2, and IL-1β mRNA. In conclusion, KRGE showed antiatherosclerotic and anti-inflammatory effects in western diet fed LDLr−/− mice and this effect could partly be mediated by RGS5 expression.

  10. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    Science.gov (United States)

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

  11. Antioxidant activity of pomegranate peel extract on lipid and protein oxidation in beef meatballs during frozen storage.

    Science.gov (United States)

    Turgut, Sebahattin Serhat; Işıkçı, Fatma; Soyer, Ayla

    2017-07-01

    Antioxidant effect of pomegranate peel extract (PE) to retard lipid and protein oxidation in beef meatballs was investigated during frozen storage at -18±1°C. Concentrated and freeze dried aqueous extract of pomegranate peel was incorporated into freshly prepared meatball mix at 0.5% and 1.0% concentrations, and compared with 0.01% butylated hydroxytoluene (BHT) and control (without any antioxidant). In PE treated samples, particularly in high PE concentration, peroxide, malondialdehyde and carbonyl formation, loss of total protein solubility and sulfhydryl groups were significantly lower than control after 6months of storage. A diminution of both myofibrillar (MP) and sarcoplasmic (SP) proteins of high molecular weight was detected after 6months of the storage according to gel electrophoresis patterns. The 1.0% PE led to maintain colour intensity (C) and hue (h°) value. The results from sensory analyses revealed that PE addition to meatballs was effective on preventing rancid odour formation. 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 oxidations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Interactions of grape tannins and wine polyphenols with a yeast protein extract, mannoproteins and β-glucan

    OpenAIRE

    Mekoue Nguela, Julie; Poncet-Legrand, Celine; Sieczkowski, N.; Vernhet, Aude

    2016-01-01

    At present, there is a great interest in enology for yeast derived products to replace aging on lees in winemaking or as an alternative for wine fining. These are yeast protein extracts (YPE), cell walls and mannoproteins. Our aim was to further understand the mechanisms that drive interactions between these components and red wine polyphenols. To this end, interactions between grape skin tannins or wine polyphenols or tannins and a YPE, a mannoprotein fraction and a β-glucan were monitored b...

  13. Electrophoretic and zymographic characterization of proteins isolated by various extraction methods from ejaculated and capacitated boar sperms

    Czech Academy of Sciences Publication Activity Database

    Zigo, Michal; Jonáková, Věra; Maňásková-Postlerová, Pavla

    2011-01-01

    Roč. 32, č. 11 (2011), s. 1309-1318 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA303/09/1285; GA MZd(CZ) NS10009; GA MŠk(CZ) 1M06011 Institutional research plan: CEZ:AV0Z50520701 Keywords : Extraction methods * Sperm proteins * Substrate zymography Subject RIV: CE - Biochemistry Impact factor: 3.303, year: 2011

  14. Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

    DEFF Research Database (Denmark)

    Callesen, Anne K; Madsen, Jonna S; Vach, Werner

    2009-01-01

    Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and ...

  15. Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

    Science.gov (United States)

    Pype, Stefan; Moechars, Dieder; Dillen, Lieve; Mercken, Marc

    2003-02-01

    Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

  16. From Extraction of Local Structures of Protein Energy Landscapes to Improved Decoy Selection in Template-Free Protein Structure Prediction.

    Science.gov (United States)

    Akhter, Nasrin; Shehu, Amarda

    2018-01-19

    Due to the essential role that the three-dimensional conformation of a protein plays in regulating interactions with molecular partners, wet and dry laboratories seek biologically-active conformations of a protein to decode its function. Computational approaches are gaining prominence due to the labor and cost demands of wet laboratory investigations. Template-free methods can now compute thousands of conformations known as decoys, but selecting native conformations from the generated decoys remains challenging. Repeatedly, research has shown that the protein energy functions whose minima are sought in the generation of decoys are unreliable indicators of nativeness. The prevalent approach ignores energy altogether and clusters decoys by conformational similarity. Complementary recent efforts design protein-specific scoring functions or train machine learning models on labeled decoys. In this paper, we show that an informative consideration of energy can be carried out under the energy landscape view. Specifically, we leverage local structures known as basins in the energy landscape probed by a template-free method. We propose and compare various strategies of basin-based decoy selection that we demonstrate are superior to clustering-based strategies. The presented results point to further directions of research for improving decoy selection, including the ability to properly consider the multiplicity of native conformations of proteins.

  17. From Extraction of Local Structures of Protein Energy Landscapes to Improved Decoy Selection in Template-Free Protein Structure Prediction

    Directory of Open Access Journals (Sweden)

    Nasrin Akhter

    2018-01-01

    Full Text Available Due to the essential role that the three-dimensional conformation of a protein plays in regulating interactions with molecular partners, wet and dry laboratories seek biologically-active conformations of a protein to decode its function. Computational approaches are gaining prominence due to the labor and cost demands of wet laboratory investigations. Template-free methods can now compute thousands of conformations known as decoys, but selecting native conformations from the generated decoys remains challenging. Repeatedly, research has shown that the protein energy functions whose minima are sought in the generation of decoys are unreliable indicators of nativeness. The prevalent approach ignores energy altogether and clusters decoys by conformational similarity. Complementary recent efforts design protein-specific scoring functions or train machine learning models on labeled decoys. In this paper, we show that an informative consideration of energy can be carried out under the energy landscape view. Specifically, we leverage local structures known as basins in the energy landscape probed by a template-free method. We propose and compare various strategies of basin-based decoy selection that we demonstrate are superior to clustering-based strategies. The presented results point to further directions of research for improving decoy selection, including the ability to properly consider the multiplicity of native conformations of proteins.

  18. Early inhibitory effects of zoledronic acid in tooth extraction sockets in dogs are negated by recombinant human bone morphogenetic protein.

    Science.gov (United States)

    Gerard, David A; Carlson, Eric R; Gotcher, Jack E; Pickett, David O

    2014-01-01

    This study was conducted with 2 purposes. The first was to determine the effect of a single dose of zoledronic acid (ZA) on the healing of a tooth extraction socket in dogs. The second was to determine if placement of recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge (ACS) - INFUSE, (Medtronic, Memphis, TN) into these extraction sockets would inhibit the inhibition on bone healing and remodeling by ZA. Nine adult female beagle dogs (2 to 3 yr old) were placed into 3 groups of 3 dogs each. Group I received 15 mL of sterile saline intravenously; group II received 2.5 mg of ZA intravenously; and group III received 5 mg of ZA intravenously. Forty-five days after treatment, all dogs underwent extraction of noncontiguous right and left mandibular first molars and second premolars. In group I, the right mandibular extraction sockets had nothing placed in them, whereas the left mandibular sockets had only ACS placed in them. In groups II and III, the right mandibular sockets had rhBMP-2/ACS placed in them, whereas the left mandibular sockets had only ACS placed. All extraction sockets were surgically closed. Tetracycline was given intravenously 5 and 12 days later, and all animals were euthanized 15 days after tooth extraction. The extraction sockets and rib and femur samples were harvested immediately after euthanasia, processed, and studied microscopically. A single dose of ZA significantly inhibited healing and bone remodeling in the area of the tooth extractions. The combination of rhBMP-2/ACS appeared to over-ride some of the bone remodeling inhibition of the ZA and increased bone fill in the extraction sites, and remodeling activity in the area was noted. The effects of rhBMP-2/ACS were confined to the area of the extraction sockets because bone activity at distant sites was not influenced. A single dose of ZA administered intravenously inhibits early healing of tooth extraction sockets and bone remodeling in this animal model. The

  19. Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

    Science.gov (United States)

    Hettenhausen, Christian; Heinrich, Maria; Baldwin, Ian T; Wu, Jianqiang

    2014-11-28

    Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

  20. Effect of acid- and alkaline-aided extractions on functional and rheological properties of proteins recovered from mechanically separated turkey meat (MSTM).

    Science.gov (United States)

    Hrynets, Yuliya; Omana, Dileep A; Xu, Yan; Betti, Mirko

    2010-09-01

    Functional and rheological characteristics of acid- and alkali-extracted proteins from mechanically separated turkey meat (MSTM) have been investigated. Extractions were carried out at 4 pH values (2.5, 3.5, 10.5, and 11.5). The study demonstrated that alkali and acid extractions resulted in significant (P hardness, chewiness, springiness, and cohesiveness) of recovered proteins were found to be unaffected (P > 0.05) by different extraction pH. The protein extracted at pH 3.5 formed a highly viscoelastic gel network as evidenced by storage modulus (G') values, whereas the gel formed from proteins extracted at pH 10.5 was found to be the weakest. The work also revealed that acid treatments were more effective for removal of total heme pigments from MSTM. Color characteristics of protein isolates were markedly improved compared to the initial material and tended to be better when subjected to acid extractions. Mechanically separated meat is one of the cheapest sources of protein obtained by grinding meat and bones together and forcing the mixture through a perforated drum. The use of mechanically separated turkey meat (MSTM) for the production of further processed poultry products is limited due to its undesirable color and textural properties. Recovery of proteins from MSTM using pH shifting process will help the poultry processors to get better returns and also create opportunity to produce functional food ingredients.

  1. The Craterostigma plantagineum glycine-rich protein CpGRP1 interacts with a cell wall-associated protein kinase 1 (CpWAK1) and accumulates in leaf cell walls during dehydration.

    Science.gov (United States)

    Giarola, Valentino; Krey, Stephanie; von den Driesch, Barbara; Bartels, Dorothea

    2016-04-01

    Craterostigma plantagineum tolerates extreme desiccation. Leaves of this plant shrink and extensively fold during dehydration and expand again during rehydration, preserving their structural integrity. Genes were analysed that may participate in the reversible folding mechanism. Analysis of transcripts abundantly expressed in desiccated leaves identified a gene putatively coding for an apoplastic glycine-rich protein (CpGRP1). We studied the expression, regulation and subcellular localization of CpGRP1 and its ability to interact with a cell wall-associated protein kinase (CpWAK1) to understand the role of CpGRP1 in the cell wall during dehydration. The CpGRP1 protein accumulates in the apoplast of desiccated leaves. Analysis of the promoter revealed that the gene expression is mainly regulated at the transcriptional level, is independent of abscisic acid (ABA) and involves a drought-responsive cis-element (DRE). CpGRP1 interacts with CpWAK1 which is down-regulated in response to dehydration. Our data suggest a role of the CpGRP1-CpWAK1 complex in dehydration-induced morphological changes in the cell wall during dehydration in C. plantagineum. Cell wall pectins and dehydration-induced pectin modifications are predicted to be involved in the activity of the CpGRP1-CpWAK1 complex. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  2. Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

    Science.gov (United States)

    Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

    2014-01-01

    Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

  3. [Suppressive Effects of Extract of Cedar Wood on Heat-induced Expression of Cellular Heat Shock Protein].

    Science.gov (United States)

    Miyakoshi, Junji; Matsubara, Eri; Narita, Eijiro; Koyama, Shin; Shimizu, Yoko; Kawai, Shuichi

    2018-01-01

     In recent years, highly antimicrobial properties of cedar heartwood essential oil against the wood-rotting fungi and pathogenic fungi have been reported in several papers. Antimicrobial properties against oral bacteria by hinokitiol contained in Thujopsis have been also extensively studied. The relation of naturally derived components and human immune system has been studied in some previous papers. In the present study, we focused on Japanese cedar, which has the widest artificial afforestation site in the country among various tree species. Extract oil was obtained from mixture of sapwood and heartwood of about 40-year cedar grown in Oguni, Kumamoto, Japan. We examined the influence of extract components from Japanese cedar woods on the expression of heat shock protein 70 (Hsp70) during heating, and on the micronucleus formation induced by the treatment of bleomycin as a DNA damaging agent. Cell lines used in this study were human fetal glial cells (SVGp12) and human glioma cells (MO54). Remarkable suppression of the Hsp70 expression induced by heating at 43°C was detected by the treatment of cedar extract in both SVGp12 and MO54 cells. We also found that cedar extract had an inhibitory tendency to reduce the micronucleus formation induced by bleomycin. From these results, the extract components from Japanese cedar woods would have an inhibitory effect of the stress response as a suppression of the heat-induced Hsp70 expression, and might have a reductive effect on carcinogenicity.

  4. Bio-oil extraction of Jatropha curcas with ionic liquid co-solvent: Fate of biomass protein.

    Science.gov (United States)

    Severa, Godwin; Edwards, Melisa; Cooney, Michael J

    2017-02-01

    The fate of oil-seed biomass protein has been tracked through all steps of a multi-phase extraction process using an ionic liquid based co-solvent system previously demonstrated to extract bio-oil and phorbol esters and to recover fermentable sugars from Jatropha oil seed. These analyses, however, did not address the fate of biomass protein. This work demonstrated that the majority of protein (∼86%) tracked with the biomass with the balance lost to co-solvent (∼12%) and methanol (∼2%) washes. A significant portion of the ionic liquid remained with the treated biomass and required aggressive methanol washes to recover. A system analysis showed a net-positive energy balance and thus the potential of this system to produce both bio-oil and protein-rich toxin-free biomass. While these results further support Jatropha as an oil seed crop, the additional costs of solvent recovery will need to be addressed if commercialization is to be realized. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Stability of Antiradical Activity of Protein Extracts and Hydrolysates from Dry-Cured Pork Loins with Probiotic Strains of LAB

    Directory of Open Access Journals (Sweden)

    Paulina Kęska

    2018-04-01

    Full Text Available The application of starter cultures to improve quality and safety has become a very common practice in the meat industry. Probiotic strains of lactic acid bacteria (LAB can also bring health benefits by releasing bioactive peptides. The aim of this work was to evaluate the stability of antiradical activity of protein extracts from LAB-inoculated dry-cured pork loins during long-term aging and evaluate their hydrolysates after simulated gastrointestinal digestion. Analyses of hydrolysates by using liquid chromatography-tandem mass spectrometry (LC-MS/MS were strengthened with in silico analysis. The highest antiradical activity of the protein extracts was observed after 180 days of aging. The influence of the strain used (LOCK, BAUER, or BB12 on the inactivation ability of ABTS radicals varied during long-term aging. The IC50 values indicated the higher antiradical properties of salt-soluble (SSF compared to water-soluble fraction (WSF of proteins. The peptides generated by in vitro digestion have MW between 700 and 4232 Da and their length ranged from 5 to 47 amino acids in a sequence where Leu, Pro, Lys, Glu, and His had the largest share. This study demonstrates that the degradation of pork muscle proteins during gastrointestinal digestion may give rise to a wide variety of peptides with antiradical properties.

  6. Comparison of salting-out and sugaring-out liquid-liquid extraction methods for the partition of 10-hydroxy-2-decenoic acid in royal jelly and their co-extracted protein content.

    Science.gov (United States)

    Tu, Xijuan; Sun, Fanyi; Wu, Siyuan; Liu, Weiyi; Gao, Zhaosheng; Huang, Shaokang; Chen, Wenbin

    2018-01-15

    Homogeneous liquid-liquid extraction (h-LLE) has been receiving considerable attention as a sample preparation method due to its simple and fast partition of compounds with a wide range of polarities. To better understand the differences between the two h-LLE extraction approaches, salting-out assisted liquid-liquid extraction (SALLE) and sugaring-out assisted liquid-liquid extraction (SULLE), have been compared for the partition of 10-hydroxy-2-decenoic acid (10-HDA) from royal jelly, and for the co-extraction of proteins. Effects of the amount of phase partition agents and the concentration of acetonitrile (ACN) on the h-LLE were discussed. Results showed that partition efficiency of 10-HDA depends on the phase ratio in both SALLE and SULLE. Though the partition triggered by NaCl and glucose is less efficient than MgSO 4 in the 50% (v/v) ACN-water mixture, their extraction yields can be improved to be similar with that in MgSO 4 SALLE by increasing the initial concentration of ACN in the ACN-water mixture. The content of co-extracted protein was correlated with water concentration in the obtained upper phase. MgSO 4 showed the largest protein co-extraction at the low concentration of salt. Glucose exhibited a large protein co-extraction in the high phase ratio condition. Furthermore, NaCl with high initial ACN concentration is recommended because it produced high extraction yield for 10-HDA and the lowest amount of co-extracted protein. These observations would be valuable for the sample preparation of royal jelly. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

    Directory of Open Access Journals (Sweden)

    Balcerkiewicz Stanislaw

    2010-12-01

    probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.

  8. Protein extraction and gel-based separation methods to analyze responses to pathogens in carnation (Dianthus caryophyllus L).

    Science.gov (United States)

    Ardila, Harold Duban; Fernández, Raquel González; Higuera, Blanca Ligia; Redondo, Inmaculada; Martínez, Sixta Tulia

    2014-01-01

    We are currently using a 2-DE-based proteomics approach to study plant responses to pathogenic fungi by using the carnation (Dianthus caryophyllus L)-Fusarium oxysporum f. sp. dianthi pathosystem. It is clear that the protocols for the first stages of a standard proteomics workflow must be optimized to each biological system and objectives of the research. The optimization procedure for the extraction and separation of proteins by 1-DE and 2-DE in the indicated system is reported. This strategy can be extrapolated to other plant-pathogen interaction systems in order to perform an evaluation of the changes in the host protein profile caused by the pathogen and to identify proteins which, at early stages, are involved or implicated in the plant defense response.

  9. Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

    Science.gov (United States)

    Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

    2011-01-01

    SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

  10. Assessment of the allergic potential of food protein extracts and proteins on oral application using the Brown Norway rat model

    NARCIS (Netherlands)

    Knippels, L.M.J.; Penninks, A.H.

    2003-01-01

    The need for widely accepted and validated animal models to test the potential allergenicity and potency of novel (biotechnology-derived) proteins has become an important issue for their safety evaluation.In this article, we summarize the results of the development of an oral sensitization protocol

  11. Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd).

    Science.gov (United States)

    Ruiz, Geraldine Avila; Xiao, Wukai; van Boekel, Martinus; Minor, Marcel; Stieger, Markus

    2016-10-15

    The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E8), 9 (E9), 10 (E10) and 11 (E11)), followed by acid precipitation. The obtained protein isolates were freeze dried. The protein isolates E8 and E9 resulted in a lower protein yield as well as less protein denaturation. These isolates also had a higher protein purity, more protein bands at higher molecular weights, and a higher protein solubility in the pH range of 3-4.5, compared to the isolates E10 and E11. Heating the 10%w/w protein isolate suspensions E8 and E9 led to increased aggregation, and semi-solid gels with a dense microstructure were formed. The isolate suspensions E10 and E11, on the other hand, aggregated less, did not form self-supporting gels and had loose particle arrangements. We conclude that extraction pH plays an important role in determining the functionality of quinoa protein isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

    Science.gov (United States)

    Li, Ao; Wang, Ruyi; Li, Xianliang; Liu, Mingyong; Fan, Jian; Guo, Kai; Luo, Bing; Chen, Tingting; Feng, Shengqiu; Wang, Yanting; Wang, Bingrui; Peng, Liangcai; Xia, Tao

    2016-05-19

    Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

  13. Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms, resulting in lipid and protein oxidative damage

    International Nuclear Information System (INIS)

    Pardo, Michal; Porat, Ziv; Rudich, Assaf; Schauer, James J.; Rudich, Yinon

    2016-01-01

    Exposure to particulate matter (PM) pollution in cities and urban canyons can be harmful to the exposed population. However, the underlying mechanisms that lead to health effects are not yet elucidated. It is postulated that exposure to repeated, small, environmentally relevant concentrations can affect lung homeostasis. This study examines the impact of repeated exposures to urban PM on mouse lungs with focus on inflammatory and oxidative stress parameters. Aqueous extracts from collected urban PM were administered to mice by 5 repeated intra-tracheal instillations (IT). Multiple exposures, led to an increase in cytokine levels in both bronchoalveolar lavage fluid and in the blood serum, indicating a systemic reaction. Lung mRNA levels of antioxidant/phase II detoxifying enzymes decreased by exposure to the PM extract, but not when metals were removed by chelation. Finally, disruption of lung tissue oxidant-inflammatory/defense balance was evidenced by increased levels of lipid and protein oxidation. Unlike response to a single IT exposure to the same dose and source of extract, multiple exposures result in lung oxidative damage and a systemic inflammatory reaction. These could be attributed to compromised capacity to activate the protective Nrf2 tissue defense system. It is suggested that water-soluble metals present in urban PM, potentially from break and tire wear, may constitute major drivers of the pulmonary and systemic responses to multiple exposure to urban PM. - Highlights: • Repeated exposure to urban PM cause systemic inflammation and oxidative damage to lung tissue lipids and proteins. • Repeated exposure to these PM extracts decreased transcription of Nrf2 protective genes. • Single as opposed to repeated exposure, induced confined lung response accompanied by activated defense mechanisms. • Metals, potentially from break and tire wear, drive the pulmonary response with exposure to urban PM. - Repeated exposures to urban PM water extracts

  14. Induction of macrophage chemotaxis by aortic extracts from patients with Marfan syndrome is related to elastin binding protein.

    Directory of Open Access Journals (Sweden)

    Gao Guo

    Full Text Available Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62.

  15. Induction of Macrophage Chemotaxis by Aortic Extracts from Patients with Marfan Syndrome Is Related to Elastin Binding Protein

    Science.gov (United States)

    Guo, Gao; Gehle, Petra; Doelken, Sandra; Martin-Ventura, José Luis; von Kodolitsch, Yskert; Hetzer, Roland; Robinson, Peter N.

    2011-01-01

    Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62). PMID:21647416

  16. Polyphenol-enriched berry extracts naturally modulate reactive proteins in model foods.

    Science.gov (United States)

    Lila, Mary Ann; Schneider, Maggie; Devlin, Amy; Plundrich, Nathalie; Laster, Scott; Foegeding, E Allen

    2017-12-13

    Healthy foods like polyphenol-rich berries and high quality edible proteins are in demand in today's functional food marketplace, but it can be difficult to formulate convenient food products with physiologically-relevant amounts of these ingredients and still maintain product quality. In part, this is because proteins can interact with other food ingredients and precipitate destabilizing events, which can disrupt food structure and diminish shelf life. Proteins in foods can also interact with human receptors to provoke adverse consequences such as allergies. When proteins and polyphenols were pre-aggregated into stable colloidal particles prior to use as ingredients, highly palatable food formulations (with reduced astringency of polyphenols) could be prepared, and the overall structural properties of food formulations were significantly improved. All of the nutritive and phytoactive benefits of the proteins and concentrated polyphenols remained highly bioavailable, but the protein molecules in the particle matrix did not self-aggregate into networks or react with other food ingredients. Both the drainage half-life (a marker of structural stability) and the yield stress (resistance to flow) of model foams made with the protein-polyphenol particles were increased in a dose-dependent manner. Of high significance in this complexation process, the reactive allergenic epitopes of certain proteins were effectively blunted by binding with polyphenols, attenuating the allergenicity of the food proteins. Porcine macrophages produced TNF-α proinflammatory cytokine when provoked with whey protein, but, this response was blocked completely when the cells were stimulated with particles that complexed whey protein with cinnamon-derived polyphenols. Cytokine and chemokine production characteristic of allergic reactions were blocked by the polyphenols, allowing for the potential creation of hypoallergenic protein-berry polyphenol enriched foods.

  17. Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.

    Science.gov (United States)

    Raveh, Avi; Schultz, Pamela J; Aschermann, Lauren; Carpenter, Colleen; Tamayo-Castillo, Giselle; Cao, Shugeng; Clardy, Jon; Neubig, Richard R; Sherman, David H; Sjögren, Benita

    2014-10-01

    Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCβ-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Saw palmetto ethanol extract inhibits adipocyte differentiation.

    Science.gov (United States)

    Villaverde, Nicole; Galvis, Adriana; Marcano, Adriana; Priestap, Horacio A; Bennett, Bradley C; Barbieri, M Alejandro

    2013-07-01

    The fruits of saw palmetto have been used for the treatment of a variety of urinary and reproductive system problems. In this study we investigated whether the fruit extracts affect in vitro adipogenesis. Saw palmetto ethanol extract inhibited the lipid droplet accumulation by induction media in a dose-dependent manner, and it also attenuated the protein expressions of C-EBPα and PPARγ. Phosphorylation of Erk1/2 and Akt1 were also decreased by saw palmetto ethanol extract. This report suggests that saw palmetto extracts selectively affect the adipocyte differentiation through the modulation of several key factors that play a critical role during adipogenesis.

  19. A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample.

    Science.gov (United States)

    Salem, Mohamed; Bernach, Michal; Bajdzienko, Krzysztof; Giavalisco, Patrick

    2017-06-01

    Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

  20. Rosemary Extracts Upregulate Nrf2, Sestrin2, and MRP2 Protein Level in Human Hepatoma HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Xiao-pei Tong

    2017-01-01

    Full Text Available In the past few decades, the incidence of liver cancer has been rapidly rising across the world. Rosemary is known to possess antioxidant activity and is used as natural antioxidant food preservative. It is proposed to have anticancer activity in treating different tumor models. In this study, we try to explore the impact of rosemary extracts on upregulating the level of Nrf2 and Nrf2-regulatory proteins, Sestrin2 and MRP2 in HepG2 cells, and to speculate its potential mechanism. The anticancer activity of rosemary extract, including its polyphenolic diterpenes carnosic acid and carnosol, was evaluated to understand the potential effect on HepG2 cells. Rosemary extract, carnosic acid, and carnosol induced the expression of Sestrin2 and MRP2 associate with enhancement of Nrf2 protein level in HepG2 cells, in which carnosic acid showed most obvious effect. Although the activation pathway of Nrf2/ARE was not exactly assessed, it can be assumed that the enhancement of expression of Sestrin2 and MRP2 may result from upregulation of Nrf2.

  1. Areca nut extract up-regulates prostaglandin production, cyclooxygenase-2 mRNA and protein expression of human oral keratinocytes.

    Science.gov (United States)

    Jeng, J H; Ho, Y S; Chan, C P; Wang, Y J; Hahn, L J; Lei, D; Hsu, C C; Chang, M C

    2000-07-01

    There are about 600 million betel quid (BQ) chewers in the world. BQ chewing is associated with increased incidence of oral cancer and submucous fibrosis. In this study, areca nut (AN) extract (200-800 microg/ml) induced the prostaglandin E(2) (PGE(2)) production by 1. 4-3.4-fold and 6-keto-PGF(1 alpha) production by 1.1-1.7-fold of gingival keratinocytes (GK), respectively, following 24 h of exposure. Exposure of GK to AN extract (>400 microg/ml) led to cell retraction and intracellular vacuoles formation. At concentrations of 800 and 1200 microg/ml, AN extract induced cell death at 21-24 and 32-52% as detected by MTT assay and cellular lactate dehydrogenase release, respectively. Interestingly, AN-induced morphological changes of GK are reversible. GK can still proliferate following exposure to AN extract. Cytotoxicity of AN extract cannot be inhibited by indomethacin (1 microM) and aspirin (50 microM), indicating that prostaglandin (PG) production is not the major factor responsible for AN cytotoxicity. PGE(2) exhibited little effect on the growth of GK at concentrations ranging from 100-1000 pg/ml. Stimulating GK production of PGs by AN extract could be due to induction of cyclooxygenase-2 (COX-2) mRNA expression and protein production. These results suggest that AN ingredients are critical in the pathogenesis of oral submucous fibrosis and oral cancer via their stimulatory effects on the PGs, COX-2 production and associated tissue inflammatory responses. AN cytotoxicity to GK is not directly mediated by COX-2 stimulation and PG production.

  2. Evaluation of the transfer and the accumulation of microcystins in tomato (Solanum lycopersicum cultivar MicroTom) tissues using a cyanobacterial extract containing microcystins and the radiolabeled microcystin-LR ("1"4C-MC-LR)

    International Nuclear Information System (INIS)

    Corbel, Sylvain; Mougin, Christian; Nélieu, Sylvie; Delarue, Ghislaine; Bouaïcha, Noureddine

    2016-01-01

    Microcystins are the most common cyanotoxins and may be expected wherever blooms of cyanobacteria occur in surface waters. Their persistence both in the irrigation water and in the soil can lead to their transfer and bioaccumulation into agricultural plants. The aim of this work was to investigate microcystin accumulation in Solanum lycopersicum cultivar MicroTom. The plant was exposed to either Microcystis aeruginosa crude extracts containing up to 100 μg eq. MC-LR L"−"1 in a soil–plant system for 90 days or pure radiolabeled "1"4C-MC-LR in a hydroponic condition for 48 h. Toxin bioaccumulation in the soil and different plant tissues was assessed both by the PP2A inhibition assay and by liquid chromatography-mass spectrometry (LC/MS/MS). After 90 days of exposure, microcystins persisted in the soil and their free extractable concentrations accumulated were very low varying between 1.6 and 3.9 μg eq. MC-LR kg"−"1 DW. Free MC-LR was detected only in roots and leaves with concentrations varying between 4.5 and 8.1 μg kg"−"1 DW and between 0.29 and 0.55 μg kg"−"1 DW, respectively. By using radioactivity ("1"4C-MC-LR), the results have reported a growing accumulation of toxins within the organs roots > leaves > stems and allowed them to confirm the absence of MC-LR in fruits after 48 h of exposure. The bioconcentration factor (BCF) was 13.6 in roots, 4.5 in leaves, and 1.4 in stems. On the other hand, the results highlight the presence of two radioactive fractions in different plant tissues. The non-extractable fraction of radioactivity, corresponding to the covalently bound MC-LR, was higher than that of the extractable fraction only in roots and leaves reaching 56% and 71% of the total accumulated toxin, respectively. Therefore, results raise that monitoring programs must monitor the presence of MCs in the irrigation water to avoid the transfer and accumulation of these toxins in crops. - Graphical abstract: Bioconcentration factors in organs of the

  3. Evaluation of the transfer and the accumulation of microcystins in tomato (Solanum lycopersicum cultivar MicroTom) tissues using a cyanobacterial extract containing microcystins and the radiolabeled microcystin-LR ({sup 14}C-MC-LR)

    Energy Technology Data Exchange (ETDEWEB)

    Corbel, Sylvain [INRA, UMR1402 ECOSYS, F-78850 Thiverval-Grignon (France); AgroParisTech, UMR1402 ECOSYS, F-78850 Thiverval-Grignon (France); Laboratoire Ecologie, Systématique et Evolution, UMR8079, Univ. Paris-Sud/CNRS/AgroParisTech, Université Paris-Sud, F-91405 Orsay (France); Mougin, Christian; Nélieu, Sylvie; Delarue, Ghislaine [INRA, UMR1402 ECOSYS, F-78850 Thiverval-Grignon (France); AgroParisTech, UMR1402 ECOSYS, F-78850 Thiverval-Grignon (France); Bouaïcha, Noureddine, E-mail: noureddine.bouaicha@u-psud.fr [Laboratoire Ecologie, Systématique et Evolution, UMR8079, Univ. Paris-Sud/CNRS/AgroParisTech, Université Paris-Sud, F-91405 Orsay (France)

    2016-01-15

    Microcystins are the most common cyanotoxins and may be expected wherever blooms of cyanobacteria occur in surface waters. Their persistence both in the irrigation water and in the soil can lead to their transfer and bioaccumulation into agricultural plants. The aim of this work was to investigate microcystin accumulation in Solanum lycopersicum cultivar MicroTom. The plant was exposed to either Microcystis aeruginosa crude extracts containing up to 100 μg eq. MC-LR L{sup −1} in a soil–plant system for 90 days or pure radiolabeled {sup 14}C-MC-LR in a hydroponic condition for 48 h. Toxin bioaccumulation in the soil and different plant tissues was assessed both by the PP2A inhibition assay and by liquid chromatography-mass spectrometry (LC/MS/MS). After 90 days of exposure, microcystins persisted in the soil and their free extractable concentrations accumulated were very low varying between 1.6 and 3.9 μg eq. MC-LR kg{sup −1} DW. Free MC-LR was detected only in roots and leaves with concentrations varying between 4.5 and 8.1 μg kg{sup −1} DW and between 0.29 and 0.55 μg kg{sup −1} DW, respectively. By using radioactivity ({sup 14}C-MC-LR), the results have reported a growing accumulation of toxins within the organs roots > leaves > stems and allowed them to confirm the absence of MC-LR in fruits after 48 h of exposure. The bioconcentration factor (BCF) was 13.6 in roots, 4.5 in leaves, and 1.4 in stems. On the other hand, the results highlight the presence of two radioactive fractions in different plant tissues. The non-extractable fraction of radioactivity, corresponding to the covalently bound MC-LR, was higher than that of the extractable fraction only in roots and leaves reaching 56% and 71% of the total accumulated toxin, respectively. Therefore, results raise that monitoring programs must monitor the presence of MCs in the irrigation water to avoid the transfer and accumulation of these toxins in crops. - Graphical abstract: Bioconcentration

  4. Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

    Science.gov (United States)

    De, Debashree; Datta Chakraborty, Piyali; Mitra, Jyotirmoy; Sharma, Kanika; Mandal, Somnath; Das, Aneesha; Chakrabarti, Saikat; Bhattacharyya, Debasish

    2013-01-01

    An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases. PMID:23555718

  5. Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    Science.gov (United States)

    Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

    2013-08-01

    Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

  6. Effect of enzymatic treatment of extracted sunflower proteins on solubility, amino acid composition, and surface activity.

    Science.gov (United States)

    Conde, José Miñones; Escobar, María del Mar Yust; Pedroche Jiménez, Justo J; Rodríguez, Francisco Millán; Rodríguez Patino, Juan M

    2005-10-05

    Industrial proteins from agriculture of either animal or vegetable origin, including their peptide derivatives, are of great importance, from the qualitative and quantitative point of view, in food formulations (emulsions and foams). A fundamental understanding of the physical, chemical, and functional properties of these proteins is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins (and their hydrolysates and peptides derivatives), are to be increasingly used in traditional and new processed food products (safe, high-quality, health foods with good nutritional value). In this contribution we have determined the main physicochemical characteristics (solubility, composition, and analysis of amino acids) of a sunflower protein isolate (SPI) and its hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis. The hydrolysates were obtained by enzymatic treatment with Alcalase 2.4 L for DH 5.62 and 23.5% and with Alcalase 2.4 L and Flavorzyme 1000 MG sequentially for DH 46.3%. The protein concentration dependence on surface pressure (surface pressure isotherm), a measure of the surface activity of the products (SPI and its hydrolysates), was obtained by tensiometry. We have observed that the degree of hydrolysis has an effect on solubility, composition, and content of the amino acids of the SPI and its hydrolysates. The superficial activity and the adsorption efficiency were also affected by the degree of hydrolysis.

  7. Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

    Science.gov (United States)

    Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

    2003-01-01

    Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

  8. Protein extraction method for the proteomic study of a Mexican traditional fermented starchy food.

    Science.gov (United States)

    Cárdenas, C; Barkla, B J; Wacher, C; Delgado-Olivares, L; Rodríguez-Sanoja, R

    2014-12-05

    Pozol is a traditional fermented maize dough prepared in southeastern Mexico. Wide varieties of microorganisms have already been isolated from this spontaneously fermented product; and include fungi, yeasts, and lactic- and non-lactic acid bacteria. Pozol presents physicochemical features different from that of other food fermentation products, such as a high starch content, in addition to a low protein content. It is these qualities that make it intractable for protein recovery and characterization. The aim of this study was to develop a methodology to optimize the recovery of proteins from the pozol dough following fermentation, by reducing the complexity of the mixture prior to 2D-PAGE analysis and sequencing, to allow the characterization of the metaproteome of the dough. The proteome of 15day fermented maize dough was characterized; proteins were separated and analyzed by mass spectrometry (LC-MS/MS). Subsequent sequence homology database searching, identified numerous bacterial and fungi proteins; with a predominance of lactic acid bacterial proteins, mainly from the Lactobacillus genus. Fungi are mainly represented by Aspergillus. For dominant genera, the most prevalent proteins belong to carbohydrate metabolism and energy production, which suggest that at 15days of fermentation not only fungi but also bacteria are metabolically active. Several methodologies have been employed to study pozol, with a specific focus toward the identification of the microbiota of this fermented maize dough, using both traditional cultivation techniques and culture independent molecular techniques. However to date, the dynamics of this complex fermentation is not well understood. With the purpose to gain further insight into the nature of the fermentation, we used proteomic technologies to identify the origin of proteins and enzymes that facilitate substrate utilization and ultimately the development of the microbiota and fermentation. In this paper we overcome the first general

  9. Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco.

    Science.gov (United States)

    Cameron, Kimberly D; Teece, Mark A; Smart, Lawrence B

    2006-01-01

    Cuticular wax deposition and composition affects drought tolerance and yield in plants. We examined the relationship between wax and dehydration stress by characterizing the leaf cuticular wax of tree tobacco (Nicotiana glauca L. Graham) grown under periodic dehydration stress. Total leaf cuticular wax load increased after each of three periods of dehydration stress using a CH2Cl2 extraction process. Overall, total wax load increased 1.5- to 2.5-fold, but composition of the wax was not altered. Homologous series of wax components were classified into organic groups; n-hentriacontane was the largest component (>75%) with alcohols and fatty acids representing drying event. Leaves excised from plants subjected to multiple drying events were more resistant to water loss compared to leaves excised from well-watered plants, indicating that there is a negative relationship between total wax load and epidermal conductance. Lipid transfer proteins (LTPs) are thought to be involved in the transfer of lipids through the extracellular matrix for the formation of cuticular wax. Using northern analysis, a 6-fold increase of tree tobacco LTP gene transcripts was observed after three drying events, providing further evidence that LTP is involved in cuticle deposition. The simplicity of wax composition and the dramatic wax bloom displayed by tree tobacco make this an excellent species in which to study the relationship between leaf wax deposition and drought tolerance.

  10. Effect of Thai saraphi flower extracts on WT1 and BCR/ABL protein ...

    African Journals Online (AJOL)

    In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. Materials and Methods: The ...

  11. Interaction of water extractable pentosans with gluten protein : effect on dough properties and gluten quality

    NARCIS (Netherlands)

    Wang, M.; Hamer, R.J.; Vliet, van T.; Oudgenoeg, G.

    2002-01-01

    The effects of modified water extractable pentosans (WEP) on gluten yield, dough properties, gluten quality and composition were studied. The results show that WEP interfere with gluten formation in both a direct and an indirect way. WEP interfere indirectly by competing for water and thus changing

  12. Effect of Pulsed Ultraviolet Light and High Hydrostatic Pressure on the Antigenicity of Almond Protein Extracts.

    Science.gov (United States)

    The efficacy of pulsed ultraviolet light (PUV) and high hydrostatic pressure (HHP) on reducing the IgE binding to the almond extracts, was studied using SDS-PAGE, Western Blot, and ELISA probed with human plasma containing IgE antibodies to almond allergens, and a polyclonal antibody against almond ...

  13. Correction of protein metabolic disorders by composite extract of Musa paradisiaca and Coccinia indica in streptozotocin-induced diabetic albino rat: an approach through the pancreas.

    Science.gov (United States)

    Mallick, Chhanda; De, Debasis; Ghosh, Debidas

    2009-04-01

    The study focused on the ability of the extracts of Musa paradisiaca and Coccinia indica on protein metabolic disorders in streptozotocin (STZ)-induced diabetes. Wistar strain rats were divided into 6 groups as control, control + composite extract treated, STZ-induced diabetes, diabetic + composite extract treated, composite extract-pretreated diabetes, and composite extract-pretreated diabetes + composite extract treated. Protein metabolic status was assessed by serum levels of urea, uric acid, albumin, and creatinine along with urine urea and albumin levels. Diabetic therapeutic ability was assessed by blood glucose, glycated hemoglobin, and serum insulin levels. Histology of the pancreas, liver, and kidney was evaluated. Indices of protein metabolic disorders were deviated from control in STZ-induced diabetes, which were protected significantly after the treatment of composite extract of M. paradisiaca and C. indica. This protection was more prominent when the extract-pretreated animals were subjected to diabetes induction by STZ. The composite extract has a protective therapeutic effect against diabetes through beta-cell regeneration capacity.

  14. Partial characterization of a novel anti-inflammatory protein from salivary gland extract of Hyalomma anatolicum anatolicum (Acari: Ixodidae ticks

    Directory of Open Access Journals (Sweden)

    Mayukh Ghosh

    2015-06-01

    Full Text Available Aim: Hyalomma anatolicum anatolicum ticks transmit Theileria annulata, causative agent of tropical theileriosis to cattle and buffaloes causing a major economic loss in terms of production and mortality in tropical countries. Ticks have evolved several immune evading strategies to circumvent hosts’ rejection and achieve engorgement. Successful feeding of ticks relies on a pharmacy of chemicals located in their complex salivary glands and secreted saliva. These chemicals in saliva could inhibit host inflammatory responses through modulating cytokine secretion and detoxifying reactive oxygen species. Therefore, the present study was aimed to characterize anti-inflammatory peptides from salivary gland extract (SGE of H. a. anatolicum ticks with a view that this information could be utilized in raising vaccines, designing synthetic peptides or peptidomimetics which can further be developed as novel therapeutics. Materials and Methods: Salivary glands were dissected out from partially fed adult female H. a. anatolicum ticks and homogenized under the ice to prepare SGE. Gel filtration chromatography was performed using Sephadex G-50 column to fractionate the crude extract. Protein was estimated in each fraction and analyzed for identification of anti-inflammatory activity. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE was run for further characterization of protein in desired fractions. Results: A novel 28 kDa protein was identified in H. a. anatolicum SGE with pronounced anti-inflammatory activity. Conclusion: Purification and partial characterization of H. a. anatolicum SGE by size-exclusion chromatography and SDSPAGE depicted a 28 kDa protein with prominent anti-inflammatory activity.

  15. Extraction, characterization, nutritional and functional properties of Roselle (Hibiscus sabdariffa Linn seed proteins

    Directory of Open Access Journals (Sweden)

    Fatoumata Tounkara

    2013-04-01

    Full Text Available Physicochemical, nutritional and functional properties of protein fractions and protein isolate (RSPI from Roselle seedwere investigated. The protein content was 91.50, 93.77, 81.55, 71.30 and 40.83% for RSPI, globulin, albumin, glutelin andprolamin, respectively. The functional properties were variable among samples. Glutelin possessed the highest water holdingcapacity and albumin the lowest. The oil holding capacity ranged from 3.47 to 7.23 mL/g and the emulsifying capacity from95 to 18 mL/g. Glutelin had the higher foam capacity, while RSPI showed the more stable foam. The molecular weight of allsamples ranged from 55,000 Da to below 14,300 Da. All the estimated nutritional parameters based on amino acids compositionsuggested that Roselle protein fractions and its isolates have good nutritional quality and could be a good source of proteinfortification for a variety of food products for protein deficient consumers as well as a potential food ingredient.

  16. Probing the molecular forces involved in binding of selected volatile flavour compounds to salt-extracted pea proteins.

    Science.gov (United States)

    Wang, Kun; Arntfield, Susan D

    2016-11-15

    Molecular interactions between heterologous classes of flavour compounds with salt-extracted pea protein isolates (PPIs) were determined using various bond disrupting agents followed by GC/MS analysis. Flavour bound by proteins decreased in the order: dibutyl disulfide>octanal>hexyl acetate>2-octanone=benzaldehyde. Benzaldehyde, 2-octanone and hexyl acetate interacted non-covalently with PPIs, whereas octanal bound PPIs via covalent and non-covalent forces. Dibutyl disulfide reacted with PPIs covalently, as its retention was not diminished by urea and guanidine hydrochloride. Using propylene glycol, H-bonding and ionic interactions were implicated for hexyl acetate, benzaldehyde, and 2-octanone. A protein-destabilising salt (Cl3CCOONa) reduced bindings for 2-octanone, hexyl acetate, and benzaldehyde; however, retention for octanal and dibutyl disulfide increased. Conversely, a protein-stabilising salt (Na2SO4) enhanced retention for benzaldehyde, 2-octanone, hexyl acetate and octanal. Formation of a volatile flavour by-product, 1-butanethiol, from dibutyl disulfide when PPIs were treated with dithiothreitol indicated occurrence of sulfhydryl-disulfide interchange reactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Avoiding acidic region streaking in two-dimensional gel electrophoresis: case study with two bacterial whole cell protein extracts.

    Science.gov (United States)

    Roy, Arnab; Varshney, Umesh; Pal, Debnath

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  18. Increased expression and local accumulation of the Prion Protein, Alzheimer Aβ peptides, superoxide dismutase 1, and Nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes

    Directory of Open Access Journals (Sweden)

    Bitel Claudine L

    2010-09-01

    Full Text Available Abstract Background Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP, and superoxide dismutase 1 (SOD1, as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies. Results Diabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2 in diabetic muscle. In contrast, we detected little change in Alzheimer Aβ precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aβ peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aβ cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aβ, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration. Conclusions The present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study

  19. Potato peel extract as a natural antioxidant in chilled storage of minced horse mackerel (Trachurus trachurus): Effect on lipid and protein oxidation

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Grejsen, Helene Drejer; Jacobsen, Charlotte

    2012-01-01

    The present work was undertaken to examine the utilisation of potato peel, a waste material, as a source of natural antioxidants for retarding lipid and protein oxidation in minced mackerel. Mackerel mince with two different concentrations (2.4 or 4.8g/kg) of water or ethanol extracts of potato...... peel and a control with no added extracts were prepared. The samples were stored at 5°C for 96h and the sampling was done at time points 0, 24, 48 and 96h. The ethanol extracts, which contained high amounts of phenolic compounds, was found to be very effective in retarding lipid and protein oxidation...

  20. Clinical and Imaging Presentation of a Patient with Beta-Propeller Protein-Associated Neurodegeneration, a Rare and Sporadic form of Neurodegeneration with Brain Iron Accumulation (NBIA).

    Science.gov (United States)

    Hattingen, Elke; Handke, Nikolaus; Cremer, Kirsten; Hoffjan, Sabine; Kukuk, Guido Matthias

    2017-12-01

    Neurodegeneration with brain iron accumulation (NBIA) is a heterogeneous group of inherited neurologic disorders with iron accumulation in the basal ganglia, which share magnetic resonance (MR) imaging characteristics, histopathologic and clinical features. According to the affected basal nuclei, clinical features include extrapyramidal movement disorders and varying degrees of intellectual disability status. The most common NBIA subtype is caused by pathogenic variants in PANK2. The hallmark of MR imaging in patients with PANK2 mutations is an eye-of-the-tiger sign in the globus pallidus. We report a 33-year-old female with a rare subtype of NBIA, called beta-propeller protein-associated neurodegeneration (BPAN) with a hitherto unknown missense variant in WDR45. She presented with BPAN's particular biphasic course of neurological symptoms and with a dominant iron accumulation in the midbrain that enclosed a spotty T2-hyperintensity.

  1. COMPARISON OF DETERGENTS FOR EXTRACTION AND ION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF SENDAI VIRUS MEMBRANE-PROTEINS

    NARCIS (Netherlands)

    WELLING, GW; FEIJLBRIEF, M; ORVELL, C; VANEDE, J; WELLINGWESTER, S

    1992-01-01

    The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e.. pentaethylene glycol monolauryl ether (C-12E5), lauryldimethylamine oxide (LDAO) and

  2. Integrated and convenient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for LC-MS/MS analysis.

    Science.gov (United States)

    Lai, Xianyin; Schneider, Bryan P

    2014-11-01

    Because fresh-frozen tissue samples associated with long-term clinical data and of rare diseases are often unobtainable at the present time, formalin-fixed paraffin-embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross-link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97-99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high-yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

    International Nuclear Information System (INIS)

    Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

    2007-01-01

    The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

  4. Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms, resulting in lipid and protein oxidative damage.

    Science.gov (United States)

    Pardo, Michal; Porat, Ziv; Rudich, Assaf; Schauer, James J; Rudich, Yinon

    2016-03-01

    Exposure to particulate matter (PM) pollution in cities and urban canyons can be harmful to the exposed population. However, the underlying mechanisms that lead to health effects are not yet elucidated. It is postulated that exposure to repeated, small, environmentally relevant concentrations can affect lung homeostasis. This study examines the impact of repeated exposures to urban PM on mouse lungs with focus on inflammatory and oxidative stress parameters. Aqueous extracts from collected urban PM were administered to mice by 5 repeated intra-tracheal instillations (IT). Multiple exposures, led to an increase in cytokine levels in both bronchoalveolar lavage fluid and in the blood serum, indicating a systemic reaction. Lung mRNA levels of antioxidant/phase II detoxifying enzymes decreased by exposure to the PM extract, but not when metals were removed by chelation. Finally, disruption of lung tissue oxidant-inflammatory/defense balance was evidenced by increased levels of lipid and protein oxidation. Unlike response to a single IT exposure to the same dose and source of extract, multiple exposures result in lung oxidative damage and a systemic inflammatory reaction. These could be attributed to compromised capacity to activate the protective Nrf2 tissue defense system. It is suggested that water-soluble metals present in urban PM, potentially from break and tire wear, may constitute major drivers of the pulmonary and systemic responses to multiple exposure to urban PM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Selective Protein Extraction from Chlorobium tepidum Chlorosomes Using Detergents. Evidence That CsmA Forms Multimers and Binds Bacteriochlorophyll a

    DEFF Research Database (Denmark)

    Bryant, Donald A; Vassilieva, Elena V; Frigaard, Niels-Ulrik

    2002-01-01

    and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters....... When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes....... Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape...

  6. Method for extraction of proteins and phosphate minerals from swine manure

    Science.gov (United States)

    The recovery of phosphorus and proteins from manure could be advantageous to both offset costs and to improve and lessen the environmental impacts of manure storage and treatment. Phosphorous in manure can contaminate rivers, lakes, and bays through runoff, if applied onto a cropland excessively. Th...

  7. Inter-laboratory optimization of protein extraction, separation, and fluorescent detection of endogenous rice allergens

    DEFF Research Database (Denmark)

    Satoh, Rie; Teshima, Reiko; Kitta, Kazumi

    2016-01-01

    In rice, several allergens have been identified such as the non-specific lipid transfer protein-1, the α-amylase/trypsin-inhibitors, the α-globulin, the 33 kDa glyoxalase I (Gly I), the 52-63 kDa globulin, and the granule-bound starch synthetase. The goal of the present study was to define optima...

  8. Biorefinery of the macroalgae Ulva lactuca: extraction of proteins and carbohydrates by mild disintegration.

    Science.gov (United States)

    Postma, P R; Cerezo-Chinarro, O; Akkerman, R J; Olivieri, G; Wijffels, R H; Brandenburg, W A; Eppink, M H M

    2018-01-01

    The effect of osmotic shock, enzymatic incubation, pulsed electric field, and high shear homogenization on the release of water-soluble proteins and carbohydrates from the green alga Ulva lactuca was investigated in this screening study. For osmotic shock, both temperature and incubation time had a significant influence on the release with an optimum at 30 °C for 24 h of incubation. For enzymatic incubation, pectinase demonstrated being the most promising enzyme for both protein and carbohydrate release. Pulsed electric field treatment was most optimal at an electric field strength of 7.5 kV cm -1 with 0.05 ms pulses and a specific energy input relative to the released protein as low as 6.6 kWh kg prot -1 . Regarding literature, this study reported the highest protein (~ 39%) and carbohydrate (~ 51%) yields of the four technologies using high shear homogenization. Additionally, an energy reduction up to 86% was achieved by applying a novel two-phase (macrostructure size reduction and cell disintegration) technique.

  9. Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death

    Energy Technology Data Exchange (ETDEWEB)

    Li, Liang, E-mail: 18710470987@163.com; Ye, Chaofei, E-mail: yechaofei001@163.com; Zhao, Rui, E-mail: 571828628@qq.com; Li, Xin, E-mail: 1458272138@qq.com; Liu, Wu-zhen, E-mail: happywuzhenliu@163.com; Wu, Feifei, E-mail: 283915941@qq.com; Yan, Jingli, E-mail: yanjingli512@163.com; Jiang, Yuan-Qing, E-mail: jiangyq@nwafu.edu.cn; Yang, Bo, E-mail: yangwl@nwafu.edu.cn

    2015-11-27

    MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death. - Highlights: • Expression of rapeseed MAPKKK4 induced ROS accumulation and cell death in leaves. • Cell death induced by MAPKKK4 is associated with membrane lipid peroxidation and DNA fragmentation. • MAPKKK4 interacts with MKK5 and MPK3. • MAPKKK4-induced ROS accumulation and cell death require downstream WIPK and SIPK. • MAPKKK4 is a novel MAPKKK modulating ROS accumulation and cell death.

  10. Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death

    International Nuclear Information System (INIS)

    Li, Liang; Ye, Chaofei; Zhao, Rui; Li, Xin; Liu, Wu-zhen; Wu, Feifei; Yan, Jingli; Jiang, Yuan-Qing; Yang, Bo

    2015-01-01

    MAPKKK is the largest family of MAPK cascade, which is known to play important roles in plant growth, development and immune responses. So far, only a few have been functionally characterized even in the model plant, Arabidopsis due to the potential functional redundancy of MAPKKK. We previously identified and cloned a few MAPKKK family genes from rapeseed. In this study, BnaMAPKKK4 was characterized as a member in eliciting accumulation of reactive oxygen species (ROS) and hypersensitive response (HR)-like cell death. This is accompanied with accumulation of malondialdehyde (MDA), anthocyanin as well as nuclear DNA fragmentation. The transcript abundance of a series of ROS accumulation, cell death, and defense response related genes were up-regulated by the expression of MAPKKK4. Further investigation identified BnaMAPKKK4 elicited ROS through the downstream MPK3. These results indicate that BnaMAPKKK4 and its downstream components function in the ROS-induced cell death. - Highlights: • Expression of rapeseed MAPKKK4 induced ROS accumulation and cell death in leaves. • Cell death induced by MAPKKK4 is associated with membrane lipid peroxidation and DNA fragmentation. • MAPKKK4 interacts with MKK5 and MPK3. • MAPKKK4-induced ROS accumulation and cell death require downstream WIPK and SIPK. • MAPKKK4 is a novel MAPKKK modulating ROS accumulation and cell death.

  11. Alcohol--Induced Polyelectrolyte-Surfactant Complex Coacervate Systems: Characterization and Applications in Enzyme and Protein Extraction

    Science.gov (United States)

    Nejati Moshtaghin, Mahboubeh

    The focus of this thesis is to achieve a better understanding of the newly discovered surfactant-polyelectrolyte complex coacervate (SPCC) systems induced by fluoroalcohol/acid as well as short chain aliphatic alcohol; and to elucidate their applications in extraction and enrichment of proteins and enzyme. We have discovered that fluoroalcohols and --acids induce complex coacervation and phase separation in the aqueous mixtures of oppositely charged anionic polyelectrolytes; specifically, sodium salts of polyacrylic acid and polymethacrylic acid and cationic surfactant (cetyltrimethylammonium bromide, CTAB) over a broad range of concentrations of mole fractions of the oppositely charged amphiphiles. Accordingly, these new classes of coacervators will significantly broaden the scope and facilitate engineering of new coacervate phases. Toward these goals, we have inspected the formation of surfactant-polyelectrolyte complex coacervates in the presence of fluoroalcohols namely hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE). Furthermore, the extent of coacervation as a function of concentrations the system components, and charge ratios of the oppositely charged amphiphiles has been investigated. Polyelectrolytes are considered to be milder reagents, as compared to surfactants, regarding proteins denaturation. This highlights the importance of a detailed investigation of the efficiency of our coacervate systems for extraction and preconcentration of proteins and enzymes, especially, when the biological activity of the extracted proteins needs to be maintained based on the objectives mentioned above, the results of the investigations have been organized in four chapters. In Chapter II, the phase behavior of the FA-SPCC will be investigated. The objective is to examine the phase behavior and phase properties with respect to the extent of coacervation in different solution conditions. In particular, the effects of different solution variables such as concentration

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

    Science.gov (United States)

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

    2011-03-01

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

  13. Proteomics of Pyrococcus furiosus (Pfu): Identification of Extracted Proteins by Three Independent Methods.

    Science.gov (United States)

    Wong, Catherine C L; Cociorva, Daniel; Miller, Christine A; Schmidt, Alexander; Monell, Craig; Aebersold, Ruedi; Yates, John R

    2013-02-01

    Pyrococcus furiosus (Pfu) is an excellent organism to generate reference samples for proteomics laboratories because of its moderately sized genome and very little sequence duplication within the genome. We demonstrated a stable and consistent method to prepare proteins in bulk that eliminates growth and preparation as a source of uncertainty in the standard. We performed several proteomic studies in different laboratories using each laboratory's specific workflow as well as separate and integrated data analysis. This study demonstrated that a Pfu whole cell lysate provides suitable protein sample complexity to not only validate proteomic methods, work flows, and benchmark new instruments but also to facilitate comparison of experimental data generated over time and across instruments or laboratories.

  14. Relationship of molecular weight distribution profile of unreduced gluten protein extracts with quality characteristics of bread.

    Science.gov (United States)

    Chaudhary, Nisha; Dangi, Priya; Khatkar, B S

    2016-11-01

    A statistical correlation was established among the molecular weight distribution patterns of unreduced gluten proteins and physicochemical, rheological and bread-making quality characteristics of wheat varieties. Size exclusion chromatography fractionated the gluten proteins apparently into five peaks. Peak I signified glutenins (30-130kDa), peak II as gliadins (20-55kDa), peak III as very low molecular weight monomeric gliadins (10-28kDa), peak IV and V, collectively, as albumins and globulins (bread loaf volume (r=0.848(∗∗)); however, peak II had negative (r=-0.818(∗∗)) impact. Bread firmness increased with increment in peak II (r=0.625(∗∗)), and decreased with accretion in peak I (r=-0.623(∗∗)). Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Immunomodulatory Activities of a Fungal Protein Extracted from Hericium erinaceus through Regulating the Gut Microbiota

    Directory of Open Access Journals (Sweden)

    Chen Diling

    2017-06-01

    Full Text Available A single-band protein (HEP3 was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities.

  16. Immunomodulatory Activities of a Fungal Protein Extracted from Hericium erinaceus through Regulating the Gut Microbiota.

    Science.gov (United States)

    Diling, Chen; Chaoqun, Zheng; Jian, Yang; Jian, Li; Jiyan, Su; Yizhen, Xie; Guoxiao, Lai

    2017-01-01

    A single-band protein (HEP3) was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities.

  17. Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis latex.

    Science.gov (United States)

    Wadeesirisak, Kanthida; Castano, Sabine; Berthelot, Karine; Vaysse, Laurent; Bonfils, Frédéric; Peruch, Frédéric; Rattanaporn, Kittipong; Liengprayoon, Siriluck; Lecomte, Sophie; Bottier, Céline

    2017-02-01

    Rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. The repartition of both proteins on the small or large rubber particles seems to differ, but their role in the irreversible coagulation of the rubber particle is still unknown. In this study we highlighted the different modes of interactions of both recombinant proteins with different classes of lipids extracted from Hevea brasiliensis latex, and defined as phospholipids (PL), glycolipids (GL) and neutral lipids (NL). We combined two biophysical methods, polarization modulated-infrared reflection adsorption spectroscopy (PM-IRRAS) and ellipsometry to elucidate their interactions with monolayers of each class of lipids. REF1 and SRPP1 interactions with native lipids are clearly different; SRPP1 interacts mostly in surface with PL, GL or NL, without modification of its structure. In contrast REF1 inserts deeply in the lipid monolayers with all lipid classes. With NL, REF1 is even able to switch from α-helice conformation to β-sheet structure, as in its aggregated form (amyloid form). Interaction between REF1 and NL may therefore have a specific role in the irreversible coagulation of rubber particles. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Independent component analysis for the extraction of reliable protein signal profiles from MALDI-TOF mass spectra.

    Science.gov (United States)

    Mantini, Dante; Petrucci, Francesca; Del Boccio, Piero; Pieragostino, Damiana; Di Nicola, Marta; Lugaresi, Alessandra; Federici, Giorgio; Sacchetta, Paolo; Di Ilio, Carmine; Urbani, Andrea

    2008-01-01

    Independent component analysis (ICA) is a signal processing technique that can be utilized to recover independent signals from a set of their linear mixtures. We propose ICA for the analysis of signals obtained from large proteomics investigations such as clinical multi-subject studies based on MALDI-TOF MS profiling. The method is validated on simulated and experimental data for demonstrating its capability of correctly extracting protein profiles from MALDI-TOF mass spectra. The comparison on peak detection with an open-source and two commercial methods shows its superior reliability in reducing the false discovery rate of protein peak masses. Moreover, the integration of ICA and statistical tests for detecting the differences in peak intensities between experimental groups allows to identify protein peaks that could be indicators of a diseased state. This data-driven approach demonstrates to be a promising tool for biomarker-discovery studies based on MALDI-TOF MS technology. The MATLAB implementation of the method described in the article and both simulated and experimental data are freely available at http://www.unich.it/proteomica/bioinf/.

  19. UTILIZATION OF MEMBRANE MICROFILTRATION IN PREPARATION OF HYDROLYZED VEGETABLE PROTEIN FROM FERMENTED RED BEAN (Phaseolus vulgaris L. EXTRACT AS FORTIFICATION AGENT

    Directory of Open Access Journals (Sweden)

    Sri Moerniati

    2010-06-01

    Full Text Available Preparation of Hydrolyzed Vegetable Protein (HVP as savory flavor from fermented red bean broth through stirred membrane cell using micro filtration membrane with pore size of 0.45 µm was performed to get fortified agent utilized in preparation of beans sauce. The objective of this work was to study an effect of pressure and kind of red bean broth extract on content of total protein, soluble protein and dry solid in the retentate and permeate as hydrolyzed vegetable protein used for fortified agent of red bean sauces. Preparation process of hydrolyzed vegetable protein was done using fixed rotary speed of 400 rpm, pressure of 20, 25 and 30 psi at room temperature. To investigate the effect of pressure on this separation, the feed were red bean broth extract fermented for 6, 8, 10 and 12 weeks, respectively. Fermentation process were conducted using salt fermentation with inoculum of Rhizopus-C1, salt and red bean ratios of 30:10:60%. The analysis of flux and contents of total protein, dissolved protein and dry solid in the retentate and permeate was carried out, and the result of experiment showed that interaction of Red bean broth extract with 6, 8, 10 and 12 weeks of fermentation and operation condition of microfiltration membrane separation tends to affect on flux and content of total protein, dissolved protein and dry solid in retentate and permeate. Red bean broth extract for 6 weeks fermentation resulted higher protein content in permeate as hydrolyzed vegetable protein than in retentate. Permeate at pressure of 25 psi gives flux value of 0.0217 mL/cm2.minute and contents of total protein of 1.31 %, dissolved protein of 6.9 mg/g, and dry solid of 2.6%, while retentate as hydrolyzed vegetable protein or fortified agent indicate contents of total protein of 1.52%, dissolved protein of 4.15 mg/g, and dry solid of 3.64%. It was found that micro filtration process was able to increase dissolved protein content of about 3 times.   Keywords

  20. Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

    Directory of Open Access Journals (Sweden)

    Xiaolin Wu

    Full Text Available Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber pr